Methods and systems for performing digital measurements

ABSTRACT

Methods, devices and systems for performing digital measurements are provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a U.S. National Phase Application under 35U.S.C. §371 of International Application No. PCT/US2012/022081, filedJan. 20, 2012, which claims the benefit of U.S. Provisional PatentApplication No. 61/434,670, filed Jan. 20, 2011, which is expresslyincorporated herein in its entirety for all purposes.

BACKGROUND OF THE INVENTION

The present invention relates to methods, devices and systems forperforming digital measurements. More specifically, the presentinvention relates to methods, devices and systems for performing digitalmeasurements in varying volumes.

Digital measurements are becoming increasingly more important inbiology, owing to their robustness, higher sensitivity and higheraccuracy that they offer. In addition, unlike analog measurements, wherethe measurement often must be calibrated with a running standard,digital measurements, based on counting of binary yes or no responses,do not require calibration and thus save user time, enhance robustnessand ease of the assay.

An important application for digital assays is the accuratequantification of DNA or RNA that is present in a sample. Here, the mostwidely used method to detect DNA or RNA is the polymerase chain reaction(PCR), where the sample is usually cycled between two or threetemperatures around 60° C. and 95° C. The use of PCR to amplify DNA orRNA has greatly advanced a wide range of disciplines, ranging from basicbiology to clinical diagnostics and forensics. One particular form ofPCR that is often used in diagnostics and biomedical research isquantitative PCR (qPCR), which not only detects the presence of DNA orRNA in the sample, but also provides an accurate measure of itsconcentration. This is an important data point for making subsequentdecisions and analysis—for example, the amount of a HIV therapeuticdrug, that is given to the patient, is determined by the amount ofdetected viral RNA load in the test sample.

So far, the most common form to carry out qPCR is real-time PCR, whichis widely used in many areas, including basic biomedical research andclinical diagnostics. In real-time PCR, the absolute concentration of asample is inferred from the time evolution of the amplification process,which is monitored with a fluorescent probe, such as a molecular beaconor Taqman® probe, that specifically recognizes the amplificationproduct. Real-time PCR is susceptible to various errors, including theformation of unwanted primer dimers, where primer molecules attach toeach other because of complementary stretches in their sequence. As aresult, a by-product is generated which competes with the target elementfor available PCR reagents, thus potentially inhibiting amplification ofthe target sequence and interfering with accurate quantification. Thequantification of target also requires the precise knowledge of theamplification efficiency for each cycle, and because the growth isexponential, tiny uncertainties in amplification efficiency (e.g. belowthe threshold detection level) will result in very large errors in thedetermination of target copy numbers. This error can become very largewhen the initial concentration of nucleic acid is low or when thefluorescent detection is not sufficiently sensitive. Despite its powerto identify and quantify target DNA from complex samples, real time PCRsuffers from the inability to quantify low sample concentrations withsufficient precision, as required for example in the detection ofpathogens or clinical diagnostics.

To overcome the difficulties of real-time PCR to quantify lowcopy-number DNA, digital or limiting dilution DNA amplification has beendeveloped, which can quantify the absolute number of template copies inthe sample more accurately. In dPCR, the total sample is divided into anarray of small volumes, such that, based on Poisson statistics, only fewvolumes contain one or more target molecules, while the majority ofvolumes contains no DNA. DNA amplification is then carried out in allvolumes simultaneously and results in an increase of fluorescence inonly those few volumes that contain target molecules. The DNA copynumber is easily and accurately determined by counting the number offluorescent volumes (i.e. those that contain a copy of DNA).

The concept of dPCR is appealing, but it is not yet widely used because(1) it can be difficult to create a large array of very small volumes(picoliters to nanoliters) used for dPCR, and (2) the dynamic range ofthe experiment is defined by the size and number of discrete arrays andis often very low. In order to accurately quantify the amount of DNA orprotein in the sample most of the compartments typically contain atmaximum one target molecule. This implies that the initial concentrationof sample be matched to the dynamic range of the assay. In other words,the initial sample concentration should be determined before inputtingthe correct concentration of sample into the device to run dPCR. Thisadds to the inconvenience of running dPCR and limits the potential ofdigital arrays with constant volumes.

Regardless of the particular reaction used, it is important to overcomethe limited dynamic range of the digital assay. A straightforward way isto extend the scope of the assay by increasing the number of digitizedvolumes of the same size. This approach is problematic; in order toaccommodate a large number of volumes, the device has to be large andwould involve fairly complex and expensive microfabrication. Thus, thereis a need for additional methods and systems for performing digitalmeasurements.

Besides the above mentioned practical issues of dPCR, widespread use ofthe method can also be impeded by precise temperature control andtemperature cycling. Generally, the temperature for the annealing andmelting step is controlled within +/−1 degree Celsius. For manyapplications, where absolute quantification of DNA and RNA is important,these factors are difficult to meet or expensive to realize, inparticular in resource-limited settings and at the point-of-care. Toprovide more ergonomic ways to amplify DNA and RNA in these settings,several isothermal methods have been developed, including rolling circleamplification (RCA), nucleic acid sequence based amplification (NASBA)or loop-mediated amplification (LAMP).

LAMP is an isothermal process for amplifying DNA or RNA with very highspecificity at a fixed temperature between 60-65 degrees Celsius. Due toits high specificity it is able to discriminate single nucleotidedifferences during amplification. As a result, LAMP has been applied forSNP (single nucleotide polymorphism) typing. LAMP has also been shown todetect viral RNA with about ten-fold higher sensitivity than RT-PCR.Another feature, that differentiates LAMP from other isothermal methods,is the ability to directly correlate the amplification of DNA with theproduction of magnesium pyrophosphate, which increases the turbidity ofthe solution. The progress of the LAMP solution can thus be followedwith a simple turbidimeter. Therefore, a non-homogeneous assay can beused for detecting the amplification products that result from LAMP. Theproduction of magnesium pyrophosphate can also be used in form of afluorescent indicator, which is particularly useful for digital assayreadout. Before the reaction, a small amount of Calcein is added to thereaction mix. During amplification, the increased production ofpyrophosphate leads to a sharp increase in Calcein fluorescence in thosevolumes that contain one or more target molecules.

These reactions proceed at a fixed temperature, which reduces instrumentcomplexity and lowers energy consumption, making them more suitable forpoint-of-care diagnostics and home-medicine devices. Translation ofthese methods into a digital format is an important step towards abetter and more accurate detection of pathogens at the point-of-care.Moreover, digital assays would also improve the accuracy of proteinamplification based assays, such as ELISA(Enzyme-Linked-Immunoadsorbent-Assay) or any single molecule basedassay, where the single molecule assay may or may not requireamplification.

In view of the above, there is a need to provide improved methods andsystems for performing digital measurements. In addition, there is alsoa need to provide additional techniques using digital measurements, suchas digital LAMP. The present invention disclosed herein provides theseneeds and more.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods, devices and systems forperforming digital measurements. More specifically, the presentinvention relates to methods, devices and systems for performing digitalmeasurements in varying volumes. In some aspects, the present inventionprovides methods and apparatus for increasing the dynamic range ofdigital measurements, including but not limited to digital PCR, digitalisothermal nucleic acid amplifications (e.g. digital NASBA and digitalLAMP), digital protein amplification (e.g. digital ELISA), digitalsingle-molecule measurements, and other forms of digital measurements.

In one aspect, the invention provides a method for increasing thedynamic range of digital measurements of a sample comprising:

creating a sample concentration gradient; and/or

creating sample volumes of different sizes.

In one embodiment, the dynamic range is increased by integrating digitalmeasurement and readout with methods and apparatus for the formation ofa concentration gradient. The concentration gradient of analytemolecules is preferably logarithmic or exponential in shape, although anumber of other shapes are possible, including but not limited tolinear, polynomial, error, Gaussian, exponential, logarithmic, and anycombinations thereof.

In another embodiment, the dynamic range is increased by using digitizedvolumes of varying sizes. For example, the dynamic range may beincreased by having on the same chip or substrate one set of array ofdigitized volumes that are 100 nL in volume, a second set of array thatis 10 nL, a third set of array that is 1 nL, a fourth set of array thatis 100 pL, a fifth set of array that is 10 pL, a sixth set of array thatis 1 pL, a seventh set of array that 100 fL, an eighth set of array thatis 10 fL, and finally a ninth set of array that 1 fL. It may not benecessary to have all these sets of array on the same chip, depending onthe final application. For some application, arrays that span 1 fL to 1nL may be sufficient; for other applications, arrays that span 100 nL to1 pL may be more appropriate. Yet for other applications, only two setsof arrays with two sets of differing volumes may be sufficient. Thenumber of sets of arrays that contain different volumes will vary, andwill depend on the size of each array. For example if a set of arraycontains one million digitized volumes, then it may not be necessary tohave arrays of 1 nL, 100 pL, 10 pL and 1 pL to span the concentrationrange of pL to nL, and simply two sets of arrays of 100 pL and 1 pL maybe sufficient.

In another aspect, the use of concentration gradients and varying sizesof digitized volumes can be combined to further increase the dynamicrange of the digital measurement. In another aspect, the invention is amethod for increasing the dynamic range of digital single-moleculemeasurements of a sample using a concentration gradient or discretevolumes of different sizes. In another aspect, the invention is aprocess of preparing a patterned surface with hydrophobic andhydrophilic patches, wherein said patches cause the formation of wetteddroplets of different size sample volumes. In another aspect, theinvention is non-transitory computer-readable medium having computerexecutable instructions stored thereon, the instructions for a machineto carry out any of the methods described herein. In another aspect, theinvention is an array comprising a patterned surface with hydrophobicand hydrophilic patches, wherein said patches cause the formation ofwetted droplets of different size sample volumes. In another aspect, theinvention is used to increase the dynamic range of digital measurementsof a sample in a non-homogeneous assay. It is important to note thatthere are a number of methods for forming concentration gradients aswell as for digitizing sample volumes. The examples provided hereinshould not be considered limiting.

This invention also describes methods and apparatus for carrying out thefollowing: (1) digital NASBA, (2) digital LAMP, (3) Performing PCR orother nucleic acid amplification using surface-attached droplet formedusing a surface patterned with hydrophobic and hydrophilic patches, and(4) Performing readout of digital nucleic acid amplification using anon-homogeneous assay.

In yet another aspect, the present invention provides a method for usingdigital measurements to determine a concentration of a sample. Themethod can include producing a first plurality of droplets having afirst volume distribution, wherein at least one of the droplets of thefirst plurality contains contents from the sample; analyzing a secondplurality of droplets having a second volume distribution to determineindividual volumes of the droplets in the second plurality and a numberof droplets in the second plurality that contain a detectable agent,wherein the first volume distribution is the same or different than thesecond volume distribution; and using individual volumes of the dropletsin the second plurality and the number of droplets in the secondplurality that contain the detectable agent to determine theconcentration of the sample.

In yet another aspect, the present invention provides a system forconducting digital measurements of a sample. The system can include asample holder containing a first plurality of droplets having a firstvolume distribution; a detector for detecting a detectable agentcontained in at least one droplet of the first plurality; and a computercomprising a memory device with executable instructions stored thereon,the instructions, when executed by a processor, cause the processor to:analyze a second plurality of droplets having a second volumedistribution to determine individual volumes of the droplets in thesecond plurality and a number of droplets in the second plurality thatcontain a detectable agent, wherein the first volume distribution is thesame or different than the second volume distribution; and useindividual volumes of the droplets in the second plurality and thenumber of droplets in the second plurality that contain the detectableagent to determine the concentration of the sample.

In yet another aspect, the present invention provides a method forperforming digital loop-mediated amplification of a sample. The methodcan include producing a plurality of droplets of the sample on amicrofluidic device, wherein at least one droplet in the pluralitycomprises a nucleic acid molecule; and performing loop-mediatedamplification in the at least one droplet to produce amplified productof the nucleic acid molecule.

For a fuller understanding of the nature and advantages of the presentinvention, reference should be had to the ensuing detailed descriptiontaken in conjunction with the accompanying drawings. The drawingsrepresent embodiments of the present invention by way of illustration.The invention is capable of modification in various respects withoutdeparting from the invention. Accordingly, the drawings/figures anddescription of these embodiments are illustrative in nature, and notrestrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the generation of digitized volumes containing a gradientconcentration of analytes. For visualization purposes, we used thefluorescent dye, fluorescein, as our analyte. We flow the gradient overan array of wells. Here, the gradient was generated using 200 μMfluorescein at 200 μL/min from a top inlet (top arrow) and water at 400μL/min from a bottom inlet (bottom arrow). Once the wells have beenfilled with the concentration gradient, we flowed light mineral oil(which is less dense than water) over the wells to create individualdigitized volumes within the wells. The wells in this example are of thesame volume, but wells of varying volumes can also be used. For thisexperiment, the wells were 75 μm in diameter and 100 μm deep and satbeneath a 2 mm tall flow chamber.

FIG. 2 depicts methods and apparatus for generating digitized volumes ofvarying sizes (volumes) using a hydrophobic and hydrophilic patternedsurface. Here, hydrophilic patches that are circular in shape arepatterned in a background of hydrophobic surface. When this surface isexposed to water (or any other aqueous solution, such as samplesolution, PCR reagent solution, or buffer) and oil, the aqueous solutionwill prefer to wet the hydrophilic patches and the oil solution willprefer to wet the hydrophobic surface. As a result, droplets (orhemi-droplets) of aqueous solution will form on the hydrophilic patchessurrounded by oil. Furthermore, the areas (e.g. area of the circle) ofthe hydrophilic patches are varied, such that the different size of thehydrophilic patch controls the volume of aqueous solution retained bythe hydrophilic patches. The volume retained by the hydrophilic patchcan have different cross sectional shapes even if the patches arecircular. The exact shape will depend on the precise hydrophobic andhydrophilic surface used as well as the nature of the oil phase and theaqueous phase. The cross section shape can be hemi-spherical or morerounded or more pancaked than hemi-spherical. The hydrophilic patchesare preferably circular, but can be a number of other shapes, includingsquare and rectangular.

FIG. 3 provides an illustration of a computer system, in accordance withan embodiment of the present invention.

FIG. 4 depicts a relationship of a cumulative number of occupieddroplets versus droplet number, in accordance with an example embodimentof the present invention. The size simulations are for concentrations(from left to right in the plot) 2.0×10⁻³, 2.0×10⁻⁴, 2.0×10⁻⁵, 2.0×10⁻⁶,2.0×10⁻⁷, and 2.0×10⁻⁸ molecules/fL. All simulations contained 10,000droplets whose diameters were drawn from a uniform distributionextending from 4 to 190 microns. All droplets were sorted by volume fromsmallest to largest and the cumulative number of occupied droplets fordroplet number i was the total number of occupied droplets with dropletnumbers ≦i.

FIG. 5 provides a simulation of cumulative number of occupied dropletsfor a concentration of 2.0×10⁻⁶ and in accordance with an exampleembodiment of the present invention. Other details are the same as inFIG. 1. In the Low region, the probability of a droplet being occupiedis very small and so the simulated curve is zero there. In the Highregion, the probability of a droplet being occupied is very close to 1and the curve rises with near unit slope there. In the Transitionregion, the probability of a droplet being occupied increases from nearzero to almost 1.

FIG. 6 shows estimated power for a uniform droplet distribution (4 to190 micron diameter), in accordance with an example embodiment of thepresent invention. The dashed lines are for measurement errors of sizeE₁ and the solid lines are for measurement errors of size E₂. The lowertwo lines are for a 1.2 fold resolution, and the upper two lines are fora 1.5 fold resolution. The dotted line is drawn at 0.95.

FIG. 7 shows estimated power for uniform droplet distribution (10 to 100micron diameter), in accordance with an example embodiment of thepresent invention. The dashed lines are for measurement errors of sizeE₁ and the solid lines are for measurement errors of size E₂. The lowertwo lines are for a 1.2 fold resolution, and the upper two lines are fora 1.5 fold resolution. The dotted line is drawn at 0.95.

FIGS. 8A-8B depict the determination of the size of emulsified dropletsusing microscopy, in accordance with an example embodiment of thepresent invention. FIG. 8A shows a dark field image of droplets on asilanized microscope slide. The size of individual droplets can bedetermined from a dark field image, if the droplets are well enoughseparated. The contrast arises from light that scatters from theinterface between aqueous phase and oil giving the droplet a circularoutline, from which the diameter of the droplet can be measured. Thescale bar represents 100 microns. FIG. 8B shows a size distribution of577 droplets measured after emulsification. The distribution isasymmetric with a median diameter of 20 μm. Most droplet diameters fallin the range between 10 and 40 microns.

FIG. 9 shows a clipped lognormal distribution used for simulations inTable 3 of the specification. The distribution is an idealizedapproximation to the experimental distribution shown in FIG. 8.

FIG. 10 depicts estimated power for clipped lognormal dropletdistribution displayed in FIG. 9. The dashed lines are for measurementerrors of size E₁ and the solid lines are for measurement errors of sizeE₂. The lower two lines are for a 1.2 fold resolution, and the upper twolines are for a 1.5 fold resolution. The dotted line is drawn at 0.95.

FIGS. 11A-11D show detection of PCR amplification in droplets, inaccordance with an example embodiment of the present invention. FIGS.11A and 11B depict images of droplets without target DNA after PCR(negative control experiment). Shown are combined dark field/ROX (11A)and dark field/FAM (11B) images, where ROX is the reference dye added tothe reaction and FAM is the fluorescence indicator used in the Taqman®probe to detect DNA amplification. The dark field illuminates thering-shaped outline of the droplet, overlaid with the Gaussian shapedfluorescence signal from the fluorophore. The insets show the intensitymeasured along the light gray line through the droplet center. Thesignal in the center region of the droplet is mostly due tofluorescence; in the absence of DNA target the center region remainsdark, the ratio between ROX and FAM fluorescence is high(ROX:FAM=120:20=6:1). FIGS. 11C and 11D are after PCR images of dropletscontaining target DNA. The combined dark field/ROX (11C) and darkfield/FAM (11D) images are shown with the corresponding line scansthrough the droplet. The line scan indicates an increased fluorescentsignal in the center region as well as a Gaussian profile that was notobserved in empty droplets. The respective ratio between the twofluorescent signals is significantly lower than for empty droplets(ROX:FAM=60:30=2:1).

FIGS. 12A-12C depict the design of the digital LAMP self-digitizationchip, in accordance with an example embodiment of the present invention.FIG. 12A is a schematic diagram showing the individual components of afully assembled chip. The microfluidic array can be embedded in a thinpiece of PDMS, which can be covered by a sealant film on top and a PDMScoated cover slip on the bottom. Air pressure can be delivered via aremovable adapter, which can be connected to an external pressuresource. FIG. 12B is an example layout of the microfluidic network. Adense array of rectangular side chambers was connected to a thin mainchannel. The whole array was surrounded by a separate water reservoir tosaturate the PDMS during incubation at 65° C. The scale bar represents 5mm. FIG. 12C shows an example geometry of the side chamber array andmain channel. All dimensions are in micrometers.

FIGS. 13A-13E show sample self-digitization in the dLAMP SD chip, inaccordance with an example embodiment of the present invention. FIG. 13Aprovides sequential images showing the initial filling of theside-chamber array with aqueous solution. After priming the chip withoil, the aqueous sample entered the main channel and distributed itselfinto the side compartments, displacing the oil phase in the chambers.FIG. 13B provides a sequence of images showing the self-digitization ofaqueous sample in the side chambers. After the whole aqueous phaseentered the chip, the tailing oil phase in the main channel isolatedindividual nanoliter sized droplets in the side chambers. FIG. 13C showsdependence of the distribution of droplet size in the side chambers onapplied pressure. The most uniform distribution with the highest averagerelative volume fraction (RVF) was obtained for an external pressure of7 psi. Lower and higher pressures resulted in formation of droplets withmore variable volumes and reduced RVF. FIG. 13D depicts a sizedistribution of RVF values for 5000 self-digitized droplets from 16individual chips. The external pressure was set to 7 psi in allexperiments. The average RVF of all droplets was 0.89±0.14. The insetshows the cumulative distribution of RVF values. The numbers correspondto the fraction of droplets with an RVF exceeding 50%, 75% and 90%,respectively. For example, 85% of all droplets filled out more than 75%of the chamber. FIG. 13E shows the reproducibility of sampleself-digitization. Shown are the average RVF value and the standarddeviation for 15 individual chips, each filled with 7-psi externalpressure.

FIGS. 14A and 14B depict chip performance at elevated temperatures, inaccordance with an example embodiment of the present invention. FIG. 14Ashows an effect of incubation on droplet volume. Typical droplet-sizedistributions are shown before and after incubation at 65° C. for 70minutes. The sample consisted of a negative control solution withouttemplate, hence no amplification was expected to occur. On average thedroplets shrank by approximately 10%. As can be seen in the inset, thedroplets located at the periphery of the array suffered from slightlyincreased shrinkage because they were more exposed to the bulk PDMS.Droplets in the center of the array were less affected by shrinkage.FIG. 14B shows a digital LAMP signature observed in our chip. A sectionof the chamber array is shown before and after incubation. The intensityprofile corresponds to a line across the centers of several chambers.Loop mediated DNA amplification in some of the chambers caused a sharpincrease in their fluorescence. Neighbouring, non LAMP-competentchambers showed no increase in fluorescence, which demonstrated thatsample crosstalk between adjacent chambers was negligible.

FIGS. 15A-15F show digital LAMP results for different DNA templateconcentrations c_(i): c_(i)=c₀/4 (15A), c_(i)=c₀/30 (15B), =c₀/150(15C), c_(i)=c₀/430 (15D) and c_(i)=c₀/1300 (15E). c₀ is theconcentration of the template stock solution. The respective fraction ofLAMP-competent chambers is analyzed in FIG. 16. For the experiment atlowest sample concentration (c₀/1300), a 535-chamber chip was used toincrease the absolute number of positive chambers. FIG. 15F shows acontrol experiment of a LAMP solution with no template added. Asexpected, none of the chambers showed an increase in fluorescence beyondbackground.

FIGS. 16A and 16B show quantification of relative and absolute changesin DNA concentration, in accordance with an example embodiment of thepresent invention. FIG. 16A shows a dilution series of the control DNAsample contained in the LoopAmp® kit, see FIG. 15. For each sampleconcentration, at least three different chips were analyzed. The solidline represents the expected fraction of positive events based on aPoisson distribution for a template concentration of 0.99×10⁴ moleculesper μl in the stock solution. This concentration was determined from the3 lowest sample. FIG. 16B shows absolute quantification of DNAconcentration. A 535 well chip is shown before and after incubation at65° C. for 70 minutes. λ-phage DNA was diluted to a final concentrationof 20 copies per μl, for which we expected approximately 12% positivechambers. After incubation we detected 9.8% LAMP-competent droplets inthe 535 well array (47 out of 479 initially formed droplets). Thedifference between expected and measured values for f_(a) may have beencaused by pipetting errors accumulated over the 8-fold dilution series.

FIG. 17 shows Table 2, which includes a comparison of confidence levelsestimated from Z-test and from simulation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and systems for performingdigital measurements. More specifically, the present invention relatesto methods and systems for performing digital measurements in varyingvolumes.

I. GENERAL

While not limiting, the present invention is based in-part on increasingthe dynamic range of a digital assay by generating volumes with variablesize. For a given sample concentration, the size of the volumes candefine the probability of being occupied by one or more molecules (e.g.,template molecules) of interest. In the example of amplification-relatedtechniques, variation of volume size can be used to alter thisoccupational probability and thus the number of wells or sample volumes(e.g., droplets) that show amplification. Notably, the present inventionis better than existing techniques that simply increase the number ofvolumes with constant size so as to increase the dynamic range. Thisadvantage is because the methods and systems disclosed herein do notrequire a large area to accommodate the volumes needed to expand dynamicrange, which, e.g., increases the likelihood of defects on the chipwhere some digitized volumes do not form properly or have other defects.In addition, simply increasing the number of volumes also increases thetime required to analyze all the digitized volumes.

Furthermore, the present invention provides methods, systems and devicesfor performing digital assays with increased dynamic range, where alarge number of volumes of varying size is generated. Unlike the abovementioned prefabricated platforms, the present invention can include useof a distribution of sizes (e.g., volumes) that is continuous ratherthan discrete. In some embodiments, droplets of variable size can becreated in various ways, either randomly or through controlledapplication of microfluidics. For example, microfluidic generation ofconstant volume droplets is well known in the art by using a T-junctionor flow-focusing device. In these systems, the size of the droplet canbe controlled by the shear rate and channel dimensions. If for a givenT-junction geometry the shear rate is continuously varied, droplets ofdifferent volumes can be generated. These methods can be realized, e.g.,by computer-controlled syringe pumps or modulated air pressure, whichadjusts the relative flow speeds of the aqueous phase and the oilcarrier fluid.

In some embodiments, droplets of various size can be generated randomly,by emulsification in a sample holder (e.g., a test tube). Dropletrandomness can simplify the experiment because, e.g., no efforts need bemade to control the size of droplets. During emulsification, droplets ofdifferent volume can be stabilized with the use of differentsurfactants. The emulsification approach is particularly useful forseveral reasons: (1) the method is compatible with basic instrumentationfound in every biomedical laboratory, (2) droplet generation is simple;it does not require complex chip design or sophisticated equipment forflow control, (3) the droplets are not confined in individual wells,which minimizes the space required to accommodate a large number ofdroplets and (4) the assay is simple because the same container can beused for droplet generation and droplet storage during amplification. Nosample transfer is needed between droplet generation and theamplification reaction.

The term “dynamic range” is defined as the ratio between the largest andsmallest possible values of a changeable quantity.

The term “digitized volumes” refers to the volumes produced afterobtaining an initial sample and separating it into physically distinctsmaller volumes in preparation for an assay.

The term “homogeneous assay” is defined where all assay components existin solution phase at the time of detection. In a homogeneous assay, nocomponent of the assay scatters detectable light.

The term “non-homogeneous assay” is defined where one or more assaycomponents are present in solid phase at the time of detection.Formation of a precipitate or particulate, such as in LAMP or rollingcircle amplification, is a common form of a heterogeneous assay. In thistype of assay, the solid phase components may scatter detectable light.

As provided herein, the term “a continuous volume distribution” isintended to describe a distribution of volumes that vary continuously,rather than by pre-defined discrete steps, across the volumedistribution. For example, chip-based platforms can include well ordroplet volumes that over a volume distribution defined by pre-defined,discrete steps fabricated as part of the chip. That is, a chip can bemade to have volumes present at 100 nL, 10 nL, and 1 nL, with no othervolumes present in between those discrete steps. In contrast, acontinuous volume distribution in not pre-defined (i.e., the volumedistribution is undefined prior to producing or forming dropletvolumes). The continuous volume distributions can, for example, beproduced via emulsification, as described further herein. In emulsions,the volumes (e.g., droplet volumes) have a discrete volume but thedroplet volumes in the distribution are undefined prior to producing thedroplets (i.e., not pre-defined by fabrication techniques) and thevolumes are randomly distributed along the continuous volumedistribution. An upper and lower boundary for droplet volumes can bemodified by the forces imparted on the emulsion (e.g., by the speed ofvortexing or the intensity of shaking). However, the droplet volumesgenerated by such techniques continuously vary along the volumedistribution produced.

In some aspects, continuous volume distributions can also becharacterized such that for any set (or plurality) of droplet volumes,its distribution function can be denoted f(x), where f(x)dx is theprobability that a given droplet in the set will have a volume between xand x+dx. (dx is an infinitesimally small number.) In certainembodiments, a continuous distribution is one where the volumes of thedroplets in the droplet set are (1) not pre-specified and (2) that forsome range x_(lower)<x<x_(upper), f(x) is always greater than zero(x_(lower) cannot be equal to x_(upper), and nothing more needs to beknown about f(x)). Thus, the present invention can in some embodimentsinclude using a droplet set drawn from a continuous distribution,measuring the volume of each droplet in the set and using the measureddroplet volumes in analysis.

I. METHODS FOR DIGITAL MEASUREMENTS

In one embodiment, the present invention provides a method for creatingconcentration gradients that are integrated with digital measurement andreadout. For example, one might integrate microfluidic gradientgeneration with a sample digitization chip. FIG. 1 shows an example ofconcentration gradients formed using microchannel inlets. For increasingthe dynamic range, a logarithmic or exponential concentration gradientis preferred, but a number of methods are now available for formingvarious types and shapes of concentration gradients on chip, includingnonlinear gradients such as power, exponential, error, Gaussian, andcubic root functions. FIG. 1 depicts the generation of digitized volumescontaining a gradient concentration of analytes.

To form a concentration gradient using the type of microfluidic designshown in FIG. 1, there needs to be only 2 inlet reservoirs or channels,but more would also be suitable for use of the invention. One inlet isused for the sample and one for buffer (or PCR reagent in the case ofdigital PCR). As the two solutions flow through the network, the samplesolution becomes diluted by the buffer (water or PCR reagent) solutionin a pre-defined fashion such that at each of the outlet channels, adifferent concentration of the sample is present. Linear, polynomial,and logarithmic gradients spanning 6 orders of magnitude have all beengenerated using variations of this design.

In another embodiment, a logarithmic or exponential gradient spanning 6orders of magnitude in concentration is used. The sample and PCRsolution is pipetted into the two inlet reservoirs, after which theywill pass over the array of wells. Once the wells have been filled withthe concentration gradient, light mineral oil or some other immisciblefluid is flowed over the wells to create individual digitized volumeswithin the wells. The wells in this example are of the same volume. Inanother embodiment of the invention, the scheme depicted in FIG. 1 isused with wells of varying volumes.

In another embodiment, the sample and PCR solution is pipetted into thetwo inlet reservoirs, after which they will pass over an array ofhydrophilic and hydrophobic patches. As the sample flows over thehydrophilic patches, they cause the formation of wetted droplets ofdifferent size sample volumes.

Alternatively, the sample can be digitized as shown using the patteredsurface shown in FIG. 2.

In another aspect of the invention, the gradients are used inconjunction with digitized volumes created using valves, wells, ordroplets. In the embodiment with droplets, the droplets can be formed ina continuous-flow fashion either in the T-channel geometry or in theflow focusing geometry, both of which are well known in the art.

To digitize the sample that had been diluted, the digitization schemedescribed in FIG. 1 can be used. Here, the sample solution containingdifferent concentrations of target molecule are flown over thetopographically patterned surface to form digitized and discrete volumesfor subsequent digital measurements and readout. Alternatively, we candigitize the sample as shown using the patterned surface shown in FIG.2.

In another embodiment, the sample can be digitized using microfluidicchannels and immiscible fluid phases. In this embodiment, the samplephase is introduced into the channel, followed by an immiscible phasewhich forms discrete sample volumes that are defined by the geometricdimensions of the side cavities (D. E. Cohen, T. Schneider, M. Wang, D.T. Chiu Anal. Chem. 82, 5707-5717).

Another aspect of the invention comprises a device for carrying out themethods of the invention. Such devices may create concentrationgradients that are integrated with digital measurement and readout. Inanother embodiment, the device carries out the method for increasing thedynamic range of digital measurements of a sample, comprising creating asample concentration gradient; and/or creating sample volumes ofdifferent sizes.

In some embodiments, the present invention includes methods to increasethe dynamic range of digital measurements that are based on creatingarrays of digitized and discrete volumes of different sizes. This methodis better than simply increasing the number of digitized volume so as toincrease dynamic range. This is because simply increasing the number ofdigitized volumes increase the area the volumes occupy as well asincrease the likelihood of having defects on the chip where somedigitized volumes do not form properly or have other defects. Simplyincreasing the number of digitized volumes also increase the timerequired to analyze all the digitized volume. A better approach toincrease the dynamic range is by creating arrays of digitized volumes ofdifferent sizes rather than simply increasing the number of digitizedvolumes. The arrays of digitized volumes of different sizes can be arandom array (e.g. droplets of different diameters all present anddistributed randomly in a container) or can be a regular array (e.g.those shown in FIG. 2).

FIG. 2 shows one example of creating arrays of digitized volumes ofdifferent sizes, where patterned surfaces are used to create arrays ofvolumes of different sizes. In this example, 7 sets of arrays arecreated, where each array contains 900 digitized volumes (30×30). Thearray is formed by creating hydrophilic circular patches in a backgroundof a hydrophobic surface. As a result, when the surface is exposed toaqueous solution and oil, the hydrophilic patches will be covered by anaqueous drop surrounded by oil. The side view of the aqueous drop isshown at the bottom of the figure. This side view depicts ahemi-spherical drop, but the shape can change (either more pancaked ormore rounded) depending on the exact surfaces we use and the oil andaqueous solution used. In one embodiment, a heavy oil is used, and thedrop will be more pancaked because the oil will push on the drop.

The circles that define each set of the 900 hydrophilic patches havedifferent sizes, ranging from 1 μm in diameter to 5 μm to 10 μm to 50 μmto 100 μm to 500 μm and finally to 1 mm in diameter. Because the volumeof the drop scales roughly as cubic to the diameter of the drop,increasing the diameter of the patch by 10 times increases the volume byabout 1,000 times. As a result, using digitized volumes of varying sizesis more efficient in terms of space and readout than simply using moredigitized volumes of the same size. In one embodiment, 900 digitizedvolumes for each set of the array is used because this number issuitable for arriving at a statistically robust digital readout.However, depending on the particular application and the neededrobustness of the readout, either more digitized volumes within each setof array or less digitized volumes can be designed.

The use of this design represents a unique scheme for creating a largearray of digitized volumes with varying sizes due to the ease of surfacepatterning hydrophilic patches of different sizes. Therefore, forapplications such as digital PCR where a wide dynamic range is oftendesired, it is highly beneficial to perform PCR in drops that arecreated using patterned surfaces as described in FIG. 2.

In some embodiments, the present invention provides methods for usingdigital measurements to determine a concentration of a sample. Themethods can include producing a first plurality of droplets having afirst volume distribution, wherein at least one of the droplets of thefirst plurality contains contents from the sample; analyzing a secondplurality of droplets having a second volume distribution to determinevolumes of the droplets in the second plurality and a number of dropletsin the second plurality that contain a detectable agent, wherein thefirst volume distribution is the same or different than the secondvolume distribution; and using volumes of the droplets in the secondplurality and the number of droplets in the second plurality thatcontain the detectable agent to determine the concentration of thesample.

In some embodiments, the volumes can be created using valves, wells, ordroplets. The embodiments that involve droplets may be particularlyuseful. Here, droplets of different volumes (diameters) can be generatedusing a wide range of methods. In one method, droplets of a definedvolume are generated using microfluidics (e.g. with T-channel or flowfocusing as well known in the art); by varying the shear rate or channeldimension, droplets of different sizes are easily formed. In anothermethod, the droplets of different volumes are generated byemulsification with the aid of different surfactants; here the dropletsof different volumes are stabilized and are controlled with the use ofdifferent surfactants. With either method, amplification of analyte(e.g. digital PCR) can be carried out simultaneously in all droplets ofdifferent volumes (sizes), after which the droplets can be flowed in asingle-file format through a flow cytometer or other similar devicewhere the size of the droplet can be determined and the fluorescencefrom the droplet can be interrogated. In this example device, thepresence of amplification product in each droplet is determined based onfluorescence and the size (volume) of each droplet is determined basedon the scattering signal from the droplet. In this way, by noting boththe size of each droplet and the presence or absence of amplificationproduct in each droplet of a given size, it is possible toback-calculate the original concentration of the analyte present in thesample after interrogating a sufficient number of droplets of differentsizes. Because the droplets are of different sizes, for a given dynamicrange, the analysis is much faster than if the droplets are all of asimilar size for reasons discussed previously.

As described herein, the volumes can be produced having a variety ofvolume distributions, which can be analyzed using a variety of differentmethods. In some embodiments, a sample can contain a molecule ormolecules of interest that can be analyzed. Discrete volumes of thesample can be generated for analysis via digital measurements. Forexample, the methods herein can include producing a plurality ofdroplets having a volume distribution. In some embodiments, theplurality of droplets of the sample can be produced in an emulsion thatincludes combining immiscible fluids, as further described herein. Inone example, a sample can include an aqueous solution that includes amolecule of interest (e.g., a nucleic acid molecule). The sample can bemixed with an oil to form droplets of the sample suspended in the oil.Depending on the method used, the volumes of the plurality of dropletsin the emulsion can be randomly distributed along a continuous volumedistribution. Furthermore, the ranges of volumes can be controlled bythe method used to form the emulsions. For example, intensity ofvortexing, shaking, and/or sonicating can be controlled to produce adesired volume distribution.

As will be appreciated by one of ordinary skill in the art, the rangesfor and volumes within a volume distribution will depend on a variety offactors for a given analysis. In some embodiments, the volumedistributions of the plurality of droplets can include a volume rangefrom about 100 nanoliters (nL) to about 1 femtoliter (fL), from about 10nL to about 10 fL, from about 1 nL to about 100 fL, from about 100 nL toabout 1 picoliter (pL), from about 10 mL to about 10 pL, from about 1 nLto about 1 pL. Depending on the selected factors for producing droplets,it is routine to define the upper and lower boundaries of a volumedistribution by, e.g., changing the intensity of mixing a sample and oilwith a surfactant. There can be ranges of volumes in the volumedistributions. For example, volumes in the distributions can range bymore than a factor of 2, by more than a factor of 10, or by more than afactor of 100 and by other factors. By ranging by a factor of 2, thelower boundary of the volume distribution can be, e.g., 10 nL with anupper boundary of 20 nL. Similarly, By ranging by a factor 10, the lowerboundary of the volume distribution can be, e.g., 10 nL with an upperboundary of 100 nL.

In addition to producing a first plurality of droplets having a firstvolume distribution, the present invention further includes analyzing asecond plurality of droplets having a second volume distribution.Analyzing the second plurality of droplets can include, e.g.,determining volumes of the droplets in the second plurality. This volumedetermination can be done using a variety of methods (e.g., usingscattering and/or microscopy). In some embodiments, individual volumesof all of the droplets in the plurality may be determined. In someembodiments, only individual volumes of some of the droplets may bedetermined. Analyzing the droplets can also include determining thenumber of droplets that include a detectable agent (i.e., one or moredetectable agents) further described herein. It is further noted thatthe second plurality of droplets is based on the same droplets producedas the first plurality of droplets. Thus, the first volume distributioncan be the same or different (e.g., narrower) than the second volumedistribution. If the distributions are the same, then each droplet inthe first plurality will be included in the second plurality. In certainembodiments, the second volume distribution is narrower than the firstvolume distribution. For example, droplets can be produced in anemulsion having a volume distribution ranging from about 1 fL to about100 nL. Depending, for example, on the concentration of the sample,analysis for digital measurements may be conducted for a volumedistribution ranging from 1 fL to about 1 nL, in which the second volumedistribution is narrower than the first volume distribution.

Reactions (e.g., amplification) can be carried out in volumes withdifferent sizes, before or during analysis of the volumes to determinein which volumes have undergone reaction (e.g., have amplified product).In certain examples, the volumes (e.g., droplets) can be sized and thenumber of occupied droplets (e.g., droplets containing a detectableagent) counted. All or just some of the droplets can be analyzed.Analysis can, for example, be achieved by flowing the droplets in asingle file through a flow cytometer or similar device, where the sizeof the droplet can be determined and the presence of amplification canbe detected. The size of the droplet can, for example, determined basedon the scattering signal from the droplet and the presence ofamplification can be indicated by a fluorescence signal from thedroplet. Alternatively, the diameter of droplets can be determined bymicroscopy. Droplets can be extracted (before, during, or aftercompletion of a reaction, e.g., amplification) from a sample holder andimaged in widefield with a CCD camera. The droplets, e.g., can be spreadout on a surface or embedded between two glass slides and placed under awidefield microscope. By using appropriate excitation and emissionfilters the fluorescence within the droplet can be quantified to revealthe presence or absence of amplification. By noting both the size of thedroplet and the presence or absence of amplification product in eachdroplet, it is possible to back-calculate the original concentration ofthe analyte present in the sample after interrogating a sufficientnumber of droplets of different sizes. Because the droplets are ofdifferent sizes, for a given dynamic range, the analysis is much fasterthan if the droplets are all of similar size. In some embodiments, themethods herein further include using a number of droplets in a pluralityand the individual volumes of the droplets in the plurality to conductdigital measurements. For example, a sample concentration of a moleculeof interest can be determined using the number of droplets in theplurality, the number of droplets in the plurality with one or moremolecules of interest, and by measuring the volume of some or all of thedroplets in the plurality. Example methods for determining sampleconcentrations can be found in the Examples section.

The present invention can be used for any technique in which digitalmeasurements provide useful information about a sample. As such, themethods, systems and devices provided herein can include a volumecontaining a detectable agent. In certain embodiments, the volume can bea well or chamber in a microfluidic chip or a droplet (e.g., a waterdroplet formed in an emulsion or on the surface of a chip) that containsthe detectable agent. It will be generally understood that thedetectable agent can include a single detectable molecule or a pluralityof detectable molecules. Other types of detectable agents can be used,e.g., beads, quantum dots, nanoparticles, and the like. Furthermore, thedetectable agent may, for example, be a molecule of interest present ina sample to be analyzed (e.g., a nucleic acid molecule in blood, serum,saliva or other solutions). Alternatively, a detectable agent can be amolecule that associates with a molecule of interest (e.g., the nucleicacid molecule) in the sample, thereby allowing the molecule to bedetected. In some embodiments, the methods and systems of the presentinvention can be used for amplification-related techniques (e.g.,digital PCR) involving digital measurements. For amplificationmeasurements, a volume (e.g., a droplet) can include a single DNAmolecule, for example, but the volume will also contain necessarycomponents that are generally well known to be used for amplificationand detection. In some embodiments, the detectable agent is fluorescentand, thus, can be detected by fluorescence-based detection methods knownin the art. However, other detection methods (e.g., absorbance,chemiluminescence, turbidity, and/or scattering) can be used to analyzethe contents of a volume. A variety of detectable agents suitable forthe present invention are generally well known in the art and can, forexample, be found in The Molecular Probes® Handbook, 11^(th) Edition(2010).

In certain embodiments, the detectable agent can be associated with amolecule of interest for detection. For example, the detectable agentcan be associated with a nucleic acid molecule (e.g., DNA or RNA), apeptide, a protein, a lipid, or other molecule (e.g., biomolecule)present in a sample. As defined herein, “associated” in the context ofthe detectable agent includes interaction with the molecule via covalentand/or non-covalent interactions. For example, the detectable agent canbe covalently attached to the molecule of interest. Alternatively, thedetectable agent can, for example, be an intercalation agent or aTaqman® probe that can be used to detect a nucleic acid molecule (e.g. aDNA and/or RNA molecule). Other detectable agents can be used, such asreference dyes that may not associate with molecules in a volume ofinterest.

Some embodiments of the present invention include producing droplets inimmiscible fluids. As is well known in the art, a wide variety ofimmiscible fluids can be combined to produce droplets of varyingvolumes. As described further herein, the fluids can be combined througha variety of ways, such as by emulsification. For example, aqueoussolution (e.g., water) can be combined with an non-aqueous fluid (e.g.,oil) to produce droplets in a sample holder or on a microfluidic chip.Aqueous solutions suitable for use in the present invention can includea water-based solution that can further include buffers, salts, andother components generally known to be used in detection assays, such asPCR. Thus, aqueous solutions described herein can include, e.g.,primers, nucleotides, and probes. Suitable non-aqueous fluids caninclude, but are not limited to, an organic phase fluid such as amineral oil (e.g., light mineral oil), a silicone oil, a fluorinated oilor fluid (e.g., a fluorinated alcohol or Fluorinert), other commerciallyavailable materials (e.g., Tegosoft®), or a combination thereof.

In addition to aqueous solutions and non-aqueous fluids, surfactants canalso be included to, e.g., improve stability of the droplets and/or tofacilitate droplet formation. Suitable surfactants can include, but arenot limited to, non-ionic surfactants, ionic surfactants, silicone-basedsurfactants, fluorinated surfactants or a combination thereof. Non-ionicsurfactants can include, for example, sorbitan monostearate (Span 60),octylphenoxyethoxyethanol (Triton X-100), polyoxyethylenesorbitanmonooleate (Tween 80) and sorbitan monooleate (Span 80). Silicone-basedsurfactants can include, for example, ABIL WE 09 surfactant. Other typesof surfactants generally well known in the art can similarly be used. Insome embodiments, the surfactant can be present at a variety ofconcentrations or ranges of concentrations, such as approximately 0.01%,0.1%, 0.25%, 0.5%, 1%, 5%, or 10% by weight.

The present invention further includes determining a concentration of asample. For example, the methods and systems can be used to determine(1) volumes of droplets and (2) a number of droplets that contain adetectable agent, which can be used to determine the concentration of asample. This information can be used in a variety of ways to determinesample concentrations. For example, target molecules are present in thesample at a concentration in units of molecules/volume. The sample canbe distributed into droplets of variable volumes that can be analyzed.The individual volumes of the droplets (all or just some) can bedetermined by methods provided herein. In addition, using detectionmethods described herein, droplets can be analyzed for containing adetectable agent or not. For a given sample concentration, some of thevariable volume droplets may contain a detectable agent and some maynot. For higher sample concentrations, generally more droplets of aplurality may contain detectable agents and vice versa; for low sampleconcentrations, fewer droplets of a plurality may be occupied by adetectable agent. As further described herein, the probabilities ofoccupancy by a detectable agent in a particular volume distribution canbe defined for a wide range of sample concentrations, which can then becompared to real data to determine the concentration of an unknownsample. Additional disclosure for determining sample concentrations canbe found in Examples 1 and 2 below. The method illustrated in theexamples involves making an initial estimate for the sampleconcentration and then calculating the number of droplets, which wouldbe predicted to contain one or more detectable agents (occupieddroplets). The estimate for the sample concentration is then adjustedusing a well-known numerical method until the predicted number ofoccupied droplets equals the actual number of occupied droplets in theplurality to within the desired degree of accuracy.

As further described herein, the present invention provides variousaspects for digital measurements that cannot be achieved by someexisting methods and systems. For example, the present invention canprovide the ability to measure sample concentration over a wide dynamicrange. In some embodiments, the dynamic range can be at least threeorders of magnitude, at least four orders of magnitude, at least fiveorders of magnitude, or at least six orders of magnitude. In someembodiments, the dynamic range can be between about 10⁻¹ to about 10⁻⁹molecules/fL, about 10⁻² to about 10⁻⁸ molecules/fL, about 10⁻³ to about10⁻⁷ molecules/fL, or about 10⁻⁴ to about 10⁻⁶ molecules/fL. In certainembodiments, determining sample concentration within a dynamic range canbe performed by detecting a detectable agent that is associated with amolecule of interest in the sample. Dynamic range can be dependent on avariety of factors, such as the range of volumes that are produced in anemulsion and/or the range of volumes that are analyzed and detected. Forexample, a first plurality of droplets having a first volumedistribution can produce a dynamic range of detectable concentrations.In some instances, the dynamic range may be decreased by analyzing anarrower volume distribution in a second plurality of droplets having asecond volume distribution. In certain embodiments, the volumedistributions include continuously varying droplet volumes.

By integrating dPCR with on-chip gradient generation, or by usingdigitized volumes of varying sizes, or the combination of both thesemethods, the invention effectively increases the dynamic range of ourdPCR chip from 1 order to 6 orders magnitude, which is comparable to thedynamic range offered by RT-PCR. By using a greater range ofconcentration gradients or arrays of digitized volumes with larger sizedifferences, the dynamic range can be increased even further if desired.This new method for carrying out quantitative PCR (qPCR) offers severalkey advantages: (1) It is more accurate as discussed previously, (2) Itobviates the need for running the type of calibration samples that isneeded for RT-PCR and thus saves time, and (3) It removes the need forreal-time sensitive fluorescence detection, which is responsible for therelatively higher cost (˜10×) of RT-PCR versus regular PCR machines.

Another aspect of the invention comprises a device for carrying out themethods of the invention. Such devices may create arrays of digitizedand discrete volumes of different sizes. In another embodiment, thedevice carries out the method for increasing the dynamic range ofdigital measurements of a sample, comprising creating a sampleconcentration gradient and creating sample volumes of different sizes.

In yet another aspect of the present invention, the methods, systems anddevices described herein can be applied to isothermal amplificationtechniques, such as digital ELISA, NASBA, and LAMP. ELISA is proteinbased and usually used for the quantification of proteins or smallmolecules. NASBA and LAMP are isothermal amplification schemes that havebeen developed to complement PCR.

In an isothermal amplification, there is no temperature cyclingoccurring as in traditional PCR. There are several types of isothermalnucleic acid amplification methods such as transcription mediatedamplification, nucleic acid sequence-based amplification, signalmediated amplification of RNA technology, strand displacementamplification, rolling circle amplification, loop-mediated isothermalamplification of DNA, isothermal multiple displacement amplification,helicase-dependent amplification, single primer isothermalamplification, and circular helicase-dependent amplification.

NASBA (Nucleic Acid Sequence Based Amplification) is an isothermal (˜40°C.) process for amplifying RNA, and has been used successfully atdetecting both viral and bacterial RNA in clinical samples. Theadvantages offered by NASBA are: (1) It has high amplificationefficiency and fast amplification kinetics, where over thousand foldamplification can be achieved within an hour or two; (2) It does notgive false positives caused by genomic dsDNA, as in the case of RT-PCR;(3) Gene expression studies can be performed without the use of intronflanking primers; (4) It does not require the degree of temperaturecontrol and feedback needed for PCR. As a result, NASBA has becomepopular for detecting viral and bacterial RNA. The fact that NASBA is anisothermal method makes it possible to run multiple samplessimultaneously with the use of a temperature controlled oven, which isan important practical advantage in many field works.

LAMP, which stands for Loop-Mediated Isothermal Amplification, iscapable of amplifying DNA with high specificity, efficiency, andrapidity under isothermal conditions (˜60° C.). Because of thecharacteristics of its amplification reaction, LAMP is able todiscriminate single nucleotide differences during amplification. As aresult, LAMP has been applied for SNP (single nucleotide polymorphism)typing. LAMP has also been shown to have about 10 fold highersensitivity then RT-PCR in the detection of viruses. Additionally,because LAMP amplification of DNA can be directly correlated with theproduction of magnesium pyrophosphate, which increases the turbidity ofsolution, the progress of LAMP has been monitored using a simpleturbidimeter. Therefore, a non-homogenous assay can be used fordetecting the amplification products that result from LAMP.

In one aspect, the present invention provides a method for performingdigital loop-mediated amplification of a sample. The method can includeproducing a plurality of droplets of the sample on a microfluidicdevice, wherein at least one droplet in the plurality comprises anucleic acid molecule (e.g., a DNA and/or a RNA molecule); andperforming loop-mediated amplification in the at least one droplet toproduce amplified product of the nucleic acid molecule. The method canalso include detecting the amplified product. In some embodiments, themethod includes determining a number of droplets in the plurality thatcomprise the amplified product; and calculating a concentration of thenucleic acid molecule in the sample using individual volumes of thedroplets in the plurality and the number of droplets in the pluralitythat contain the nucleic acid molecule. The microfluidic device caninclude a plurality of chambers configured to form the plurality ofdroplets. Additional aspects of performing digital LAMP with the presentinvention can be found in Example 3.

Despite some of the advantages offered by NASBA and LAMP, one importantdrawback is the difficulty with performing quantification, which wouldbe beneficial in most situations. Quantification often requiresmeticulous calibration and control using standards amplified underidentical conditions, which can be very tedious (especially for fieldstudies) and is not practical in many cases. For non-homogenous assays,such as the detection of precipitate in LAMP, accurate calibration canbe especially challenging.

Rolling circle amplification (RCA) is an isothermal nucleic-acidamplification method. It differs from the polymerase chain reaction andother nucleic-acid amplification schemes in several respects. DuringRCA, a short DNA probe anneals to a target DNA of interest, such as theDNA of a pathogenic organism or a human gene containing a deleteriousmutation. The probe then acts as a primer for a Rolling CircleAmplification reaction. The free end of the probe anneals to a smallcircular DNA template. A DNA polymerase is added to extend the primer.The DNA polymerase extends the primer continuously around the circularDNA template generating a long DNA product that consists of manyrepeated copies of the circle. By the end of the reaction, thepolymerase generates many thousands of copies of the circular template,with the chain of copies tethered to the original target DNA. Thisallows for spatial resolution of target and rapid amplification of thesignal. The use of forward and reverse primers can change the abovelinear amplification reaction into an exponential mode that can generateup to 1012 copies in 1 hour. The calibration required for suchquantitative measurements can be cumbersome.

To overcome this drawback, the present invention provides digitalisothermal amplifications, such as NASBA and LAMP, where the use of anarray of digitized volumes, similar to digital PCR, is used for carryingout digital NASBA, digital LAMP, and rolling circle amplification.Furthermore, by using concentration gradients and/or arrays of digitizedvolumes of different sizes, we can effectively increase the dynamicrange of these digital measurements. The current method ideallycomplements these isothermal amplification schemes to make them aquantitative technique for measuring the presence of RNA and DNA. Inanother embodiment of the invention, the method is applied to antibodybased amplification. In another embodiment, the method is applied tospecific molecule recognition based amplification.

III. SYSTEMS FOR DIGITAL MEASUREMENTS

In yet another aspect, the present invention provides systems for usingdigital measurements to determine a concentration of a sample. Thesystems can include a sample holder containing a first plurality ofdroplets having a first volume distribution; a detector for detecting adetectable agent contained in at least one droplet of the firstplurality; and a computer comprising a memory device with executableinstructions stored thereon, the instructions, when executed by aprocessor, cause the processor to: analyze a second plurality ofdroplets having a second volume distribution to determine volumes of thedroplets in the second plurality and a number of droplets in the secondplurality that contain a detectable agent, wherein the first volumedistribution is the same or different than the second volumedistribution; and use volumes of the droplets in the second pluralityand the number of droplets in the second plurality that contain thedetectable agent to determine the concentration of the sample. In someembodiments, the concentration of the detectable agent in the sample isused to calculate the sample concentration.

As described further herein, the volumes used for digital measurementscan be generated and analyzed by a variety of ways. The presentinvention includes a sample holder that can be used to hold the volumesso that the volumes can be further processed and/or analyzed. The sampleholders of the present invention can include test tubes, eppendorftubes, arrays of wells on a microarray or in a microfluidic chip, amicrofluidic chip configured to generate droplets, as well as othercommercially available or otherwise generally known devices capable ofholding discrete volumes (e.g., wells or droplets) of a sample. Thesystems of the present invention further include a detection systemconfigured to analyze the volumes. The detection systems can includedetectors for analyzing the contents of the volumes, determining volumesof droplets, and/or other characteristics of interest. The methodsdescribed herein will be generally compatible with any known systemscapable of detecting and analyzing volumes (e.g., droplets and/orwells).

In yet another aspect, the systems can include a computer-readablestorage medium for conducting digital measurements. Thecomputer-readable storage medium has stored thereon instructions that,when executed by one or more processors of a computer, cause thecomputer to: analyze a second plurality of droplets having a secondvolume distribution to determine a number of droplets in the secondplurality that contain the detectable agent, wherein the first pluralityof droplets comprises the second plurality of droplets and the secondvolume distribution is narrower than the first volume distribution; anduse the number of droplets in the second plurality, the volumes of someor all of the droplets in the second plurality and the number ofdroplets in the second plurality containing one or more detectableagents to determine a concentration of the detectable agent in thesample.

In yet another aspect, a system is provided for analyzing volumes todetect and calculate information from the analyzed volumes. The systemincludes one or more processors, and a memory device includinginstructions executable by the one or more processors. When theinstructions are executed by the one or more processors, the system atleast receives a user input to analyze volumes (e.g., a plurality ofdroplets). The system can be configured to carry out aspects of themethods of the present invention, such as counting a number of volumes(e.g., droplets), determining volumes of a plurality of droplets in avolume distribution and use the number of the droplets containing one ormore detectable agents to determine a concentration of the detectableagent in the sample. The system also provides data to a user. The dataprovided to the user can include the concentration of the detectableagent in the sample or a sample concentration.

FIG. 3 is a simplified block diagram of a computer system 100 that maybe used for the methods, media and systems described herein. In variousembodiments, computer system 100 may be used to implement any of thesystems or methods illustrated and described above. As shown in FIG. 3,computer system 100 includes a processor 102 that communicates with anumber of peripheral subsystems via a bus subsystem 104. Theseperipheral subsystems may include a storage subsystem 106, comprising amemory subsystem 108 and a file storage subsystem 110, user interfaceinput devices 112, user interface output devices 114, and a networkinterface subsystem 116.

Bus subsystem 104 provides a mechanism for enabling the variouscomponents and subsystems of computer system 100 to communicate witheach other as intended. Although bus subsystem 104 is shownschematically as a single bus, alternative embodiments of the bussubsystem may utilize multiple busses.

Network interface subsystem 116 provides an interface to other computersystems and networks. Network interface subsystem 116 serves as aninterface for receiving data from and transmitting data to other systemsfrom computer system 100. For example, network interface subsystem 116may enable a user computer to connect to the Internet and facilitatecommunications using the Internet.

User interface input devices 112 may include a keyboard, pointingdevices such as a mouse, trackball, touchpad, or graphics tablet, ascanner, a barcode scanner, a touch screen incorporated into thedisplay, audio input devices such as voice recognition systems,microphones, and other types of input devices. In general, use of theterm “input device” is intended to include all possible types of devicesand mechanisms for inputting information to computer system 100.

User interface output devices 114 may include a display subsystem, aprinter, a fax machine, or non-visual displays such as audio outputdevices, etc. The display subsystem may be a cathode ray tube (CRT), aflat-panel device such as a liquid crystal display (LCD), or aprojection device. In general, use of the term “output device” isintended to include all possible types of devices and mechanisms foroutputting information from computer system 100. An advertisement may beoutput by computer system 100 using one or more of user interface outputdevices 114.

Storage subsystem 106 provides a computer-readable storage medium forstoring the basic programming and data constructs. Software (programs,code modules, instructions) that when executed by a processor providethe functionality of the methods and systems described herein may bestored in storage subsystem 106. These software modules or instructionsmay be executed by processor(s) 102. Storage subsystem 106 may alsoprovide a repository for storing data used in accordance with thepresent invention. Storage subsystem 106 may include memory subsystem108 and file/disk storage subsystem 110.

Memory subsystem 108 may include a number of memories including a mainrandom access memory (RAM) 118 for storage of instructions and dataduring program execution and a read only memory (ROM) 120 in which fixedinstructions are stored. File storage subsystem 110 provides anon-transitory persistent (non-volatile) storage for program and datafiles, and may include a hard disk drive, a floppy disk drive along withassociated removable media, a Compact Disk Read Only Memory (CD-ROM)drive, an optical drive, removable media cartridges, and other likestorage media.

Computer system 100 can be of various types including a personalcomputer, a portable computer, a workstation, a network computer, amainframe, a kiosk, a server or any other data processing system. Due tothe ever-changing nature of computers and networks, the description ofcomputer system 100 depicted in FIG. 3 is intended only as a specificexample for purposes of illustrating the embodiment of the computersystem. Many other configurations having more or fewer components thanthe system depicted in FIG. 3 are possible.

The specific dimensions of any of the apparatuses, devices, systems, andcomponents thereof, of the present invention can be readily varieddepending upon the intended application, as will be apparent to those ofskill in the art in view of the disclosure herein. Moreover, it isunderstood that the examples and embodiments described herein are forillustrative purposes only and that various modifications or changes inlight thereof may be suggested to persons skilled in the art and areincluded within the spirit and purview of this application and scope ofthe appended claims. Numerous different combinations of embodimentsdescribed herein are possible, and such combinations are considered partof the present invention. In addition, all features discussed inconnection with any one embodiment herein can be readily adapted for usein other embodiments herein. The use of different terms or referencenumerals for similar features in different embodiments does notnecessarily imply differences other than those expressly set forth.Accordingly, the present invention is intended to be described solely byreference to the appended claims, and not limited to the embodimentsdisclosed herein.

IV. EXAMPLES Example 1 Example Theoretical Framework

This example describes a non-limiting theoretical framework that can beused to describe certain aspects of the present invention describedherein. It will be understood that the method described in this exampleis one of many ways to determine concentrations of sample using thepresent invention. Under this theoretical framework, it is assumed thatthe target molecules are present in the sample at a concentration C_(S)in units of molecules/volume. The sample is distributed into digitalvolumes of variable volumes; the distribution of target molecules intothe droplets follows Poisson statistics. For each droplet in the digitalarray and as shown in Equation 1, the average number of targets dependson its volume V_(i), and the initial sample concentration C_(S):

$\begin{matrix}{{P\left( {n,{C_{S}V_{i}}} \right)} = {\frac{\left( {C_{S}V_{i}} \right)^{n}}{n!}{\exp\left( {{- C_{S}}V_{i}} \right)}}} & (1)\end{matrix}$is the probability of finding n molecules in a droplet of volume V_(i)for a given concentration C_(S) of target molecules in solution. Theamplification reaction will cause a droplet containing one or moremolecules to be distinguishable from empty droplets, e.g. by itsfluorescence intensity. In this method we know only whether a droplet isempty or occupied. The associated probabilities are shown in Equation 2:P(0,C _(S) V _(i))=exp(−C _(S) V _(i))P(n>0,C _(S) V _(i))=1−exp(−C _(S) V _(i))  (2)

In order to determine the concentration C_(S) of target molecules,P(n>0, C_(S)V_(i)) needs to be summed over a sufficiently large numberof droplets with respective volumes V_(i).

Simulation Methods

Simulations of a digital amplification assay are described, e.g. digitalPCR, digital LAMP or digital ELISA, with continuous variable volumes tovalidate, that the target concentration C_(S) can be accuratelydetermined.

For each simulation a fixed number of droplets, N_(d), and simulatedconcentration of analyte molecules, C_(S), was chosen. Simulations wereperformed for various values of C_(S). A random number generator wasused to choose the diameters for N_(d) droplets from a given range anddistribution. To illustrate the principle of this invention, simulationswere performed assuming that the droplet diameter D is uniformlydistributed between a minimum diameter of 4 microns and a maximumdiameter of 190 microns. This results in simulated volumes which rangefrom 34 to 3.6×10⁶ fL. A more realistic distribution of dropletdiameters is discussed below.

In each simulation a random number generator is used to determinewhether a particular simulated droplet is occupied or empty, when theinitial target concentration is C_(S). The total number of occupieddroplets in a simulation, N_(S), is counted and subsequently compared tothe expected number of occupied droplets

$\begin{matrix}{N_{E} = {\sum\limits_{i = 1}^{N_{d}}\left( {1 - {\exp\left( {{- C}\; V_{i}} \right)}} \right)}} & (3)\end{matrix}$where the most probable value of N_(E) is obtained for C=C_(S). Usingthe volumes of the N_(d) droplets, equation (3) can be fit to N_(S) toobtain a best fit value of the concentration, with C being the onlyadjustable parameter. We use the Newton-Rhapson algorithm to find thezero of N_(S)−N_(E). The initial value of C is obtained by replacing theV_(i)'s in equation (3) with the median volume of the distribution andsolving for C. The algorithm then typically takes 5-11 iterations forthe changes in C to fall below 1 part in 10⁵. The value of C at thatpoint is taken to be the best fit value of C.

For the simulations, some of the goals are (i) to determine howaccurately this procedure is in estimating C_(S) and (ii) if twodifferent samples yield different best fit values of C, how to calculatethe confidence that the samples have different concentrations.

This method includes the measurement of the volume of each droplet, andthere can be an error associated with that which is taken into account.To simulate this, a Gaussian distributed error is added to each of thesimulated diameter to yield simulated measured diameters. Thesesimulated measured diameters are then used to calculate simulatedmeasured volumes, {circumflex over (V)}_(i) which are substituted intoequation (3) to yield the expected number of occupied droplets based onmeasured volumes.

$\begin{matrix}{{\hat{N}}_{E} = {\sum\limits_{i = 1}^{N_{d}}\left( {1 - {\exp\left( {{- C}\;{\hat{V}}_{i}} \right)}} \right)}} & (4)\end{matrix}$

One method for determining the droplet diameters is by microscopy. Theaccuracy of this method can depend on the numerical aperture (NA) of theobjective used as well as other details of the imaging system. Forimaging a large number of stationary droplets on a surface the NA wouldlikely be less than 1 and in our experience the errors in the diametermeasurements is typically 0.5 to 1.0 microns independent of the size ofthe droplet. We simulated two different magnitudes of measurement error,denoted E₁ and E₂. For E₁ the standard deviation of the Gaussiandistributed error added to a droplet diameter is the larger of 1 micronor 8% of that droplet's diameter. The relative error is included so thatthe simulation includes a non-negligible measurement error for thelargest droplets. For E₂ the standard deviation of the Gaussiandistributed error added to a droplet diameter is the larger of 2 micronor 15% of that droplet's diameter. In both cases there is one limitplaced on the simulated measured diameters. If a particular Gaussiandeviate results in a droplet diameter being less than 0.5 microns, thenthat deviate is discarded and a new one generated for that droplet.

In the analysis of an experiment C_(S) is the actual, unknownconcentration which the procedure is attempting to determine, C is thebest fit estimate, the V_(i)'s are actual volumes of the droplets andthe {circumflex over (V)}_(i)'s are the volumes measured by theexperimenter.

Simulation Results

Results for a typical set of simulations are shown in FIG. 4.Simulations were performed with 10,000 droplets and six differentconcentrations C_(S) (2.0×10⁻³, 2.0×10⁻⁴, 2.0×10⁻⁵, 2.0×10⁻⁶, 2.0×10⁻⁷,and 2.0×10⁻⁸ molec/fL). The droplet sizes were drawn from a uniformdistribution of diameters between 4.0 and 190 microns. For eachsimulation, the droplets were sorted by volume, the droplet with thesmallest volume was number 1 and the droplet with the largest volume wasnumber 10,000. FIG. 4 plots the cumulative number of occupied droplets.The cumulative number occupied for the ith droplet is the sum of theoccupied droplets for all droplets ≦i.

The shape of the curve is a function of both the concentration and theshape of the distribution of droplet volumes. A distribution which isuniform in droplet volume (instead of droplet diameter) would yielddifferent shaped curves, as would non-uniform distributions. For anygiven distribution of sizes, curves of the type in FIG. 4 will risemonotonically with droplet number, and curves for differentconcentrations will never cross. So once the distribution of dropletsizes has been measured, the shape of the curve and the total number ofoccupied droplets depends only on the concentration of analytemolecules. For sufficiently large concentrations, there is a dropletvolume where the probability of being occupied is very close to 1.0.Once the droplet number corresponding to that volume has been reached,the curve rises with near unit slope for all larger droplet numbers. Forsufficiently small concentrations there is a droplet volume below whichthe probability of being occupied is indistinguishable from 0.0. For alldroplet numbers smaller than the droplet number corresponding to thatvolume the curves equal zero.

Effect of Measurement Error of the Droplet Diameters on Best Fit Results

As described herein, the present invention involves individuallymeasuring sizes of droplets. Since the volume of a droplet enters intothe probability of it being occupied, it would appear that it isimportant that the volumes be measured very accurately. For the range ofdroplet sizes in this simulation, the smallest droplets 4 microndiameter) are not substantially larger than typical errors in measuringdroplet diameters by light microscopy (≈1 micron standard deviation).The resulting relative errors in the droplet volume can be significantand might be expected to degrade the accuracy of this method.Surprisingly, this does not appear to be the case. Table 1 compares theresults of fitting equation (3) with the results of fitting equation (4)to a series of simulations. For each concentration, 100 simulations wereperformed, each containing 2000 droplets. For equation (3) the simulatedvolumes, V_(i), were used. For equation (4), simulated measured volumes,{circumflex over (V)}_(i), were used. The point is that the results offitting the simulated results using equation (3) represents the best fitconcentrations that are obtained when the volumes of the droplets areknown with perfect accuracy, while the results of fitting with equation(4) includes the effect of errors in the measurement of the dropletvolumes. The average and standard deviations for the best fitconcentrations using equations (3) and (4) are listed in Table 1 forboth E₁ and E₂.

TABLE 1 Effects of volume measurement errors in simulations of 2000droplets Simulation Best fit using V Best fit using {circumflex over(V)} C_(S) C σ_(C) C σ_(C) E₁ (±1.0 micron or 8% error(a)) 8.00 × 10⁻⁹7.80 × 10⁻⁹ 2.04 × 10⁻⁹ 7.66 × 10⁻⁹ 2.01 × 10⁻⁹ 8.00 × 10⁻⁸ 7.97 × 10⁻⁸6.78 × 10⁻⁹ 7.85 × 10⁻⁸ 6.73 × 10⁻⁹ 8.00 × 10⁻⁷ 8.02 × 10⁻⁷ 3.38 × 10⁻⁸8.10 × 10⁻⁷ 3.52 × 10⁻⁸ 8.00 × 10⁻⁶ 8.01 × 10⁻⁶ 4.45 × 10⁻⁷ 8.17 × 10⁻⁶4.59 × 10⁻⁷ 8.00 × 10⁻⁵ 8.06 × 10⁻⁵ 6.98 × 10⁻⁶ 8.22 × 10⁻⁵ 7.17 × 10⁻⁶8.00 × 10⁻⁴ 8.11 × 10⁻⁴ 9.64 × 10⁻⁵ 8.23 × 10⁻⁴ 1.00 × 10⁻⁴ 8.00 × 10⁻³8.19 × 10⁻³ 1.55 × 10⁻³ 8.66 × 10⁻³ 1.84 × 10⁻³ E₂ (±2.0 micron or 15%error(a)) 8.00 × 10⁻⁹ 8.03 × 10⁻⁹ 2.13 × 10⁻⁹ 7.53 × 10⁻⁹ 2.01 × 10⁻⁹8.00 × 10⁻⁸ 8.00 × 10⁻⁸ 6.89 × 10⁻⁹ 7.61 × 10⁻⁸ 6.76 × 10⁻⁹ 8.00 × 10⁻⁷8.02 × 10⁻⁷ 3.35 × 10⁻⁸ 8.30 × 10⁻⁷ 3.77 × 10⁻⁸ 8.00 × 10⁻⁶ 8.00 × 10⁻⁶4.57 × 10⁻⁷ 8.60 × 10⁻⁶ 5.13 × 10⁻⁷ 8.00 × 10⁻⁵ 8.04 × 10⁻⁵ 6.76 × 10⁻⁶8.62 × 10⁻⁵ 7.46 × 10⁻⁶ 8.00 × 10⁻⁴ 8.10 × 10⁻⁴ 1.06 × 10⁻⁴ 8.52 × 10⁻⁴1.16 × 10⁻⁴ 8.00 × 10⁻³ 8.15 × 10⁻³ 1.51 × 10⁻³ 1.01 × 10⁻² 2.93 × 10⁻³(a)The larger of the fixed and relative errors for each droplet is usedas the standard deviation of the Gaussian distributed error added tothat droplet to generated simulated measured diameters from thesimulated diameters. These are used to calculate the simulated measuredvolumes ({circumflex over (V)}) and simulated volumes (V), respectively.

The differences between the best fit concentrations using equations (3)and (4) are very small except the very largest concentrations. Even whenthe error is ±2 microns or 15%, the error only reaches 25% for thelargest concentration. Furthermore, for each concentration, the standarddeviations of the best fit results using equations (3) and (4) are,except for the largest concentration, nearly the same, without anyapparent systematic difference between them. This suggests that for mostof the concentrations in Table 1 that the largest source of variabilityin the best fit concentrations is the variation in the droplet occupancynumbers from one simulation to the next. Any variability in the best fitresults due to the droplet size measurement errors has a small ornegligible effect on the concentration determination so long as it isunbiased. All of this is despite the fact that for the smallest dropletsone standard deviation of the error is close to half the diameter forthe simulations with 2.0 micron error (E₂). This is an important resultsince it means that this method does not require that an unreasonablyhigh accuracy in the measurement of the droplet diameters. As isdiscussed below it primarily requires that any measurement error beunbiased. This surprising result can be explained with the help of FIG.5.

FIG. 5 shows the results of a simulation of the cumulative number ofoccupied droplets for a concentration of 2.0×10⁻⁶. The simulationincludes 10,000 droplets whose diameters are uniformly distributedbetween 4 and 190 microns. The curve is divided into three regions: Low,Transition and High. In the Low region, the probability of a dropletbeing occupied is very small and so the simulated curve is zero there.In the High region, the probability of a droplet being occupied is veryclose to 1 and the curve rises with near unit slope there. In theTransition region the probability of a droplet being occupied increasesfrom near zero to almost 1. Measurement errors for droplets within theLow or High regions will have little or no effect on the total number ofoccupied droplets. From equation (4) we see that a measurement error ona droplet within the Low region simply replaces a term in the sum whichis nearly zero with a different term which is also nearly zero whichshould result in a negligible change in the total number of occupieddroplets. A similar effect occurs in the High region where measurementerrors replace terms in equation (4) which are nearly 1.0 with otherterms which are also nearly 1.0. If the errors are unbiased, as they arein the simulations in Table 1, then there will also be some cancellationof the errors. The terms in sum in equation (4) which correspond todroplets in either the Low or High region are as likely to be replacedby a slightly larger term as they are to be replaced by a slightlysmaller term. Therefore, there will be some cancellation of the dropletvolume measurement errors which will reduce the effect of those errorson the best fit concentration. If the errors are unbiased and if thereare enough droplets distributed throughout the volumes which define theTransition region, then some cancellation of the volume measurementerrors for droplets within the Transition region also appears to occur.In fact it seems that volume measurement errors will introducesignificant errors in the best fit concentrations only when themeasurement errors significantly change the shape of the distributionfunction of the volumes, and even then only if the change occurs forthose droplets within the Transition region. The effect of the Gaussiandistributed measurement errors used in Table 1 on the distribution ofdiameters is only noticeable near the ends of the distribution. Forexample, the smallest droplets generated in these simulations havediameters near 4 microns. However, when measurement error is included,the simulated measured diameters can be less than 4 microns. That is,the distribution of simulated measured diameters will extend to smallersizes than the actual simulated diameters, which would be a slightbroadening of the distribution function near 4 microns. For thesesmallest droplets, the probability of being occupied is calculated usingthe simulated size, but when fitting the data, the probability of beingoccupied is calculated using the smaller measured size. This can lead tothe fit over estimating the concentration when the Transition regionincludes these smallest droplets. This is what is seen in Table 1,though the effect is not very large. There should be an equivalenteffect at the lowest concentrations due to a slight broadening of thedistribution function near 190 microns, but the effect is not asnoticeable.

The range of droplet diameters used in these simulations was chosen sothat any reasonable sized droplet volume measurement error might beexpected to have a noticeable effect on attempts to estimate theconcentration from the total number of occupied droplets. However, thisdoes not appear to have occurred, even though the range ofconcentrations simulated (10⁶) is larger than the range of volumessimulated (10⁵). Other simulations with droplet diameters uniformlydistributed over smaller ranges (10 and 10³) yield similar results inthat reasonably accurate best fit concentrations can be obtained over arange of simulated concentrations larger than the range of volumes byapproximately a factor of 10.

A different question arises if this method is used to determine if theconcentrations of two different samples are different. For that theconfidence and statistical power of this method must be consider. Thisis done in the next section.

Estimation of Confidence and Power

The ability of a method to distinguish a difference in concentrationscan be described in terms of confidence and power. (Lieber, R. L. (1990)“Statistical Significance and Statistical Power in Hypothesis Testing”,J. Orthopaedic Research 8, 304-309) If measurements on two differentsamples yield two different best fit concentrations (C₁ and C₂), itwould be useful to know how confident one could be in asserting that theconcentrations of the two samples are different, and also theprobability of being wrong if one concluded that they were notdifferent. The risk of a false positive result (Type I error) iscontrolled by requiring that the results have a required minimumconfidence and the risk of a false negative result (Type II error) iscontrolled by requiring that the method have a required power.

For the comparison of two best fit concentrations from two differentsamples, the null hypothesis would be that the two samples had the same(unknown) concentration. If α is the probability that two samples withthe same concentration could result in best fit concentrations whichdiffer in magnitude by more than |C₁−C₂|, then (1−α) is the confidenceassociated with rejecting the null hypothesis. The acceptable minimumvalue of (1−α) is chosen to limit false positive results. The use of thepower to guard against false negatives is described later.

One method used to estimate confidence levels is the Z-test.

$\begin{matrix}{Z = \frac{C_{1} - C_{2}}{\sqrt{\sigma_{1}^{2} + \sigma_{2}^{2}}}} & (5)\end{matrix}$A confidence level of 95% is a common choice and requires Z>1.96. TheC_(n) the best fit results of equation (4) and σ_(n) ² is the varianceassociated with the measurement of C_(n). A tractable analyticalexpression for the σ_(n) ² does not appear to exist for our method, soit is necessary to estimate the σ_(n) ² by simulation. Furthermore, oneadditional limitation of the Z test is that it assumes that themeasurement results are normally distributed. Since this is notnecessarily the case a second simulation method to estimate theconfidence was performed and compared with the Z test results.Hereafter, the two methods will be denoted the Z method and Pairs or Pmethod.

For the Z method estimate of the confidence, we used two differentvalues of C_(S) (C_(S1)=2.0×10⁻⁵ and C_(S2)=2.2×10′). For each value ofC_(S) a set of 5000 droplets was randomly selected from a uniformdistribution in droplet diameters extending from 4 to 190 microns andequation (2) was used to simulate the number of occupied droplets. TheE₁ size of measurement errors was used to generate simulated measuredvolumes. Best fit values for each set were determined to obtain C₁. andC₂, where C_(n), is the best fit result for the simulation which usedC_(Sn) for its simulated concentration. For each C_(n), additional Zmethod simulations were performed. C_(n) and the simulated measuredvolumes, {circumflex over (V)}_(i), were used in equation (2) to obtainN_(Z)=1000 additional, simulated total number of occupied droplets forthe set of {circumflex over (V)}_(i). These are then fit using equation(2) to obtain N_(Z) best fit concentrations, C_(n) ^(Z). The standarddeviation of the C_(n) ^(Z) is used for σ_(n) in equation (5). Thiscorresponds to the information an experimenter would have. An estimateof the confidence could be calculated from the best fit concentrations,C_(n), and the measured droplet volumes {circumflex over (V)}_(i).

For the Pairs method estimate of the confidence we assume that the nullhypothesis is that the two best fit results (C₁ and C₂) were eachobtained from a sample whose actual concentration, C, is the average ofthe two best fit concentrations. N_(p)=500 additional sets of dropletdiameters were simulated and for each, the number of occupied dropletswere simulated for the concentration C, and the E₁ size of measurementerrors was used to generate simulated measured volumes. A best fitconcentration C′ was obtained for each set. Then M_(p)=500 pairs ofvalues were randomly selected with replacement from the set of C′ andthe absolute value of their differences (δC′_(p), p=1, . . . , M_(p))were compared with ΔC=C₂−C₁. The fraction of the δC′_(p) which aregreater than ΔC provides an estimate of α, the probability that twomeasurements made from a sample with concentration C would differ fromeach other by more than ΔC. The P method estimate of α is used tocalculate the confidence, which equals (1−α).

The entire procedure for obtaining two estimates of the confidence wasrepeated for pair of C_(S) values 100 times. For each repetition, a newset of simulated volumes was generated for each C_(S) and equation (2)was used to generate the number of occupied droplets for each C_(S).Simulated measured volumes were generated and used in equation (4) tofit the results and obtain a new pair of best fit concentrations (C₁ andC₂). The Z method and P methods were then used to estimate theconfidence.

The small difference between the two chosen values of C_(S) (10%)results in confidences which range from near zero to near 1. Thispermits us to compare the two methods of estimating the confidence overa range of values, and reasonable agreement was obtained in all cases.Table 2 in FIG. 17 shows a few of the 100 results. In particular, Table2 shows the 5 sets which resulted in the smallest values of theconfidence, 6 sets which resulted in confidence values at the median,and 5 sets which resulted in the confidence values near 1.0. The resultsfrom the Z method are shown in the first 5 columns and the results fromthe P method are shown in the last 3 columns. The values of theconfidence, (1−α), for the two different methods are in columns 5 and 8and reasonable agreement between them is found for all of the setssimulated. The absence of an analytical expression does not pose aproblem as, the Z test as implemented in the Z method appears to besufficiently accurate. While we continue to use the P method in thecalculation of the power, in practice the Z method is a reasonablealternative and is computationally less expensive. Furthermore, the Pmethod requires a numerical method for generating distributions of thedroplet sizes which is a good approximation to the actual distribution,while the Z method does not. This last requirement is not a problem insimulations, but could be a difficulty in analyzing experimentalresults, so the Z method would be more useful in that case. We note thatthe Z method, which uses the same experimentally measured droplet sizesfor all of its N_(Z) simulations, produces confidences which are inreasonable agreement with those of the P method, which generates a newset of simulated droplet volumes and associated measurement errors foreach of its N_(P) simulations. This implies that for a sufficientlylarge number of droplets, the variation in a particular sample'sdistribution of droplet volumes is a less significant contributor to thevariability in the number of occupied droplets than Poisson probabilityof a droplet being occupied (equation (2)).

The confidence level provides an estimate of how likely a resultindicating two samples have different concentrations could be a falsepositive. The power provides an estimate of how likely false negativesare. For this the procedures for the previous set of simulations wereused. For a given percentage difference between the reference and testconcentrations the fraction of results which are too close together tosatisfy a required confidence level is calculated. This is the fractionof results (β) which would yield false negatives for the particularconcentration difference and required confidence level (in this case95%) and the statistical power is (1−β). The results for this test arein FIGS. 6-7. The different figures are the resulting power for 2different distributions of droplet diameters and include both the E₁ andE₂ measurement errors. FIGS. 6 and 7 are the results for uniformdistributions with diameter ranges of 4 to 190 microns (FIG. 6, volumerange ≈10⁵), and 10 to 100 microns (FIG. 7, volume range ≈10³). Eachconcentration on the abscissa is tested against concentrations which are20% and 50% larger (1.2 fold and 1.5 fold resolution, respectively). Theresults in all of the figures are for sets of 5000 droplets and theother parameters are the same as used in the pairs method for estimatingthe confidence.

When distinguishing between samples whose concentrations differ by 20%this method is capable of powers >0.95 for a range of concentrationswhich span a factor of ≈100. If the requirement is relaxed from 1.2 foldto 1.5 fold resolution, the range of concentrations for which the poweris >0.95 is spans more than a factor of 10⁴ for the broad uniformdistribution (FIG. 6, droplet diameters ranging from 4 to 190 microns)and almost 10⁴ for the other one (FIG. 7, droplet diameters ranging from10 to 100 microns). The location of that range depends on the shape ofthe droplet size distribution. The range for which the power is >0.95increases if the required resolution is relaxed or the number ofdroplets is increased.

The total volume of sample in a 5,000 droplet sample for the uniformdistribution ranging from 4 to 190 microns is approximately 5 uL. Thetotal number of copies in the 8×10′ simulation is approximately 35/5000droplets, or about 7800 copies/ml. Smaller concentrations can be reachedusing a range of droplets which include larger sized droplets or largernumbers of droplets.

Example 2 Experimental Method for the Generation of Droplets withVariable Volume and Determining their Size

Droplets of variable size can be created in various ways. In one methodmicrofluidics can be used to generate droplets of various sizes at aT-junction or flow-focusing device. This uses specialized chips intowhich the two components (oil phase and aqueous sample) have to beloaded first, before the aqueous sample becomes digitized by dropletformation. In this example, we employed a simpler, more user-friendlymethod in which droplets of various sizes are generated randomly, namelyby emulsification in a test tube or otherwise suitable container. In thefirst embodiment, the aqueous phase containing the reaction mix ispipetted into a 0.2 mL PCR test tube that is prefilled with anappropriate oil-surfactant mix. As provided herein, the oil phaseconsisted of 73% Tegosoft®, 20% light mineral oil and 7% ABIL WE 09surfactant, which were freshly mixed and equilibrated for at least 30minutes before use. Emulsions formed with this mixture tend to showsuperior thermostability during standard emulsion PCR. After pipettingthe aqueous phase to the oil mix, droplets of variable size were formedby vortexing for about ten seconds at about 3000 rpm. Emulsification canbe further enhanced by adding a small stir bar to the mix, which duringthe vortexing promotes breakup of the aqueous phase into smallerdroplets. The presence of the surfactant in the oil stabilizes theemulsion, such that the individual aqueous droplets did not fuse. Inanother embodiment, aqueous phase and oil mix were added to a smallcollection microtube, containing a small stainless steel bead. The tubewas subsequently shaken at 15-17 Hz for 20 seconds to generate theemulsion. The emulsion was then transferred into a 0.2 mL PCR test tubefor running the PCR reaction.

In order to measure the distribution of droplet sizes, a small volume ofthe emulsion was spread out on a silanized glass slide and covered withan excess volume of the oil-surfactant mix. The sample was then placedunder a widefield microscope and illuminated from the bottom with darkfield illumination. In this scheme, an opaque disc of suitable dimensionis placed in the light path of the illumination source, blocking outmost of the light before the specimen. As a result only light from anouter ring of illumination reaches the specimen. The position of theopaque disc is matched to the microscope objective such, that only asmall portion of light that is scattered from the specimen can enter theobjective lens. Since the sample contrast comes from light that isscattered from the sample, the resulting image is dominated by the ringshaped circumference of the individual droplets as shown in FIG. 8A. Dueto the large contrast obtained in dark field, the droplet diameter canbe easily determined for each circle in the image, provided the dropletsare sparsely enough distributed.

FIG. 8B shows the distribution of 577 droplets after emulsification. Thedistribution is highly asymmetric with a median diameter of 20 μm. Thisemulsification preferentially generated smaller droplets around 10-40μm, where the smallest droplets had a diameter of about 6-8 μm, and thelargest droplet diameter found was of the order of 70-80 μm. This factorof 10 in droplet diameter translates into a dynamic volume range ofroughly 1000.

As demonstrated in FIG. 8 any real process used to generate the varyingsizes of droplets is more likely to produce a peaked rather than auniform distribution. To verify, that our conclusions derived from auniform distribution of droplet sizes remain valid for a peakeddistribution like that obtained in the experiment, we simulated digitalPCR experiments with droplets, whose size distribution is shown in FIG.9. The distribution is a lognormal distribution in which only diametersbetween 8 and 64 microns are included. This distribution was used in thesame type of simulations performed for Table 1, and the results arelisted in Table 3.

TABLE 3 Effects of volume measurement errors in simulations of 2000droplets with non uniform distribution Simulation Best lit using V Bestfit using {circumflex over (V)} C_(S) C σ_(C) C σ_(C) E₁ (±1.0 micron or8% error(a)) 2.00 × 10⁷   2.01 × 10⁷   6.14 × 10⁸   1.97 × 10⁷   6.03 ×10⁸   2.00 × 10⁻⁶ 2.01 × 10⁻⁶ 2.13 × 10⁻⁷ 1.97 × 10⁻⁶ 2.11 × 10⁻⁷ 2.00 ×10⁻⁵ 2.00 × 10⁻⁵ 8.89 × 10⁻⁷ 2.01 × 10⁻⁵ 9.11 × 10⁻⁷ 2.00 × 10⁻⁴ 2.00 ×10⁻⁴ 8.76 × 10⁻⁶ 2.04 × 10⁻⁴ 9.10 × 10⁻⁶ 2.00 × 10⁻³ 2.01 × 10⁻³ 1.46 ×10⁻⁴ 2.09 × 10⁻³ 1.59 × 10⁻⁴ E₂ (±2.0 micron or 15% error(a)) 2.00 ×10⁻⁷ 2.00 × 10⁻⁷ 6.16 × 10⁻⁸ 1.87 × 10⁻⁷ 5.79 × 10⁻⁸ 2.00 × 10⁻⁶ 1.99 ×10⁻⁶ 2.13 × 10⁻⁷ 1.89 × 10⁻⁶ 2.06 × 10⁻⁷ 2.00 × 10⁻⁵ 2.00 × 10⁻⁵ 8.76 ×10⁻⁷ 2.01 × 10⁻⁵ 9.39 × 10⁻⁷ 2.00 × 10⁻⁴ 2.00 × 10⁻⁴ 8.73 × 10⁻⁶ 2.13 ×10⁻⁴ 9.95 × 10⁻⁶ 2.00 × 10⁻³ 2.02 × 10⁻³ 1.44 × 10⁻⁴ 2.36 × 10⁻³ 2.00 ×10⁻⁴ (a)The larger of the fixed and relative errors for each droplet isused as the standard deviation of the Gaussian distributed error addedto that droplet to generated simulated measured diameters from thesimulated diameters. These are used to calculate the simulated measuredvolumes ({circumflex over (V)}) and simulated volumes (V), respectively.

The results in Table 3 suggest that it even with an asymmetricdistribution of modest dynamic range it is possible to obtain reasonablyaccurate (±10%) estimates of the analyte molecule concentrations overfairly substantial concentration ranges. This is important, since itshows that a simple process like emulsification, which can be performedin almost all laboratory settings, can produce a large enoughdistribution of droplets to accurately determine target DNAconcentrations over 4-5 orders of magnitude. Small refinements canimprove this dynamic range even further.

As a further test we estimated the power of the method for clippedLogNormal function that is displayed in FIG. 10 (volume range ≈500). Asin the case of FIGS. 6 and 7, four sets of simulations were performedspanning the concentration range from 8×10⁻⁹ to 8×10⁻³ molecules/fL. Thedata show that the power that can be achieved with the experimentaldistribution of droplets is comparable to that estimated for the case ofuniformly distributed droplets of similar range (FIG. 7). The simple anduser-friendly procedure of generating droplets in emulsion, combinedwith the robust performance of the analysis should make this inventionappealing as a new method to perform digital assays with increaseddynamic range.

Analysis of Amplification after dPCR

In order to visualize the presence of amplification product within adroplet, a fluorescence probe is added into the reaction mix thatspecifically recognizes the presence of the amplicon. In most cases thisis either a molecular beacon, i.e. a hairpin structure, whosefluorescence is highly quenched in its closed conformation and whoseintensity is increased once it hybridizes to amplified target DNA, or aTaqman® probe, which hybridizes to the target DNA, and leading tocleavage of a fluorescent reporter from the probe DNA during the nextamplification step.

These probes have a non-negligible background fluorescence and therelative increase in intensity during amplification can be rather small,depending of the amount of probe added to the reaction. Furthermore, theexcitation intensity might vary across the field of view during thedetection process. A simple intensity measure can thus sometimes lead toambiguous results, since the data usually has to be thresholded todetermine whether the change in fluorescence is significant to indicatethe generation of amplification product. In all our experiments wetherefore add a small amount (1-2 μM) of the red fluorescent dyeRhodamine as a reference dye into the reaction mix, whose spectralsignature can be well separated from the fluorescence probe that reportson amplification. We then build the ratio of two intensities, onemeasured from the reference dye and one measured from the probe. Theintensity ratio will not be affected by changes in droplet volume due tofusion or shrinkage, or unwanted changes in the excitation power, sincethis would affect both dyes in the same way, but leaving their ratiounchanged. FIG. 11 shows images for two PCR reactions, where (1) thedroplets contained no target DNA during amplification (negative control)and (2) the reaction mix contained target DNA. Fluorescence images thatshow the presence of the reference dye (ROX) and probe (FAM), were takenin epifluorescence, while the droplets were simultaneously imaged indark field through bottom illumination. The insets show the measuredintensity across a line through the center of the droplets. In theabsence of target DNA (A,B) the intensity in the FAM channel isdominated by the dark field signal from the droplets, with the centerportion showing no increase in signal, while in the ROX channel the darkfield signal superimposes on a Gaussian shaped fluorescence signal fromthe reference dye. The ratio between the intensity from the centerregion in the ROX and FAM channel is large (ROX:FAM=120:20=6:1). In thepresence of DNA in the reaction mix (C,D) an increased signal in thedroplet center is also observed in the FAM channel, indicating thatamplification took place in that droplet. Consequently, the ratiobetween ROX and FAM fluorescence is significantly smaller,ROX:FAM=60:30=2:1.

One other way in which this invention could be implemented is withdifferent groups of droplets containing the sample solution at differentdilutions. The droplets containing the sample at different dilutionswould have to be kept separate from each other. If that could be done,then the only change to the analysis of the experiment would be thereplacement of factors of the volume in the equations by d_(i)V_(i),where d_(i) is the dilution factor for the ith droplet. For instance,equation (2) would becomeP(0,C _(S) d _(i) V _(i))=exp(−C _(S) d _(i) V _(i))P(n>0,C _(S) d _(i) V _(i))=1−exp(−C _(S) d _(i) V _(i))  (6)with similar changes for the other equations. This would permit theextension of the method to accurately measure larger concentrations thanthe droplet size distribution would otherwise be capable of.

Example 3 Example of Digital Isothermal DNA Amplification

This examples describes a method that does not only apply to PCR-basedDNA amplification, but also is suitable for every kind of digital assaygenerally known in the art. The simplicity and robustness of the methodmakes this example particularly useful at the point-of-care and inresource-limited settings, where the availability of specializedbenchtop equipment might be limited.

Automated Sample Self-Digitization with Reproducible Droplet Formation

The digital LAMP chip was designed as a series of rectangular cavitiesto hold the droplets, which were positioned along a smaller rectangularchannel used for sample delivery (FIG. 12). The chip layout was designedto improve droplet stability and retention, automation of the fillingprocess, and operation at elevated temperatures. A reduction of theheight ratio between main channel and side cavities to ⅓ reduced thechance of crosstalk between chambers. The depth of the side chamber wasextended to 400 μm, which we found to improve droplet retention. Airpressure regulated flow was used to ensure reproducibility androbustness of the automated filling. Evaporation of the aqueous dropletsat higher temperatures was minimised by various measures: a) Thechambers were arranged in a dense array and embedded between only a thintop and bottom layer of PDMS. b) An additional sacrificial water channelwas placed around the array. c) A self-adhesive film was added on top ofthe chip as a vapour barrier.

Prior to loading the aqueous phase, the chip was primed with thecontinuous phase (light mineral oil with 0.025% w/w SPAN-80 surfactant).Fresh LAMP solution was introduced into the inlets and self-digitizedinto nanoliter-sized droplets inside the side chambers. FIGS. 13A and13B show a sequence of images taken during chamber filling and dropletformation. The aqueous phase entered the main channel and slowlydisplaced the oil from the side chambers. Once the whole sample enteredthe chip, the tailing oil phase sheared off the fluids at the opening ofthe side compartments and isolated the nanoliter droplets.

The uniformity of droplet size across the array depended on the appliedair pressure as shown in FIG. 13C. The volume of the retained dropletswas estimated from images taken before chip incubation as described inthe experimental section. In our setup, an air pressure between 7-8 psiyielded the most uniform distribution of droplet size, where themajority of droplets showed near complete volume retention, indicated bya relative volume fraction (RVF) near unity. Only a fraction of smallersizes was found, most of which near the outlet where initialside-chamber filling by the aqueous phase was incomplete. Higher orlower pressures caused the RVF distribution to broaden, with moredroplets showing significantly reduced RVF values.

FIG. 13D shows a cumulative histogram of 5000 droplets. More than 96% ofdroplets occupied at least half the chamber volume, while for 56% of alldroplets, the RVF exceeded 90%. On average, we found a RVF value of0.89±0.14. Droplet uniformity was desirable, since it reduced errors inquantification due to difference in initial droplet volume. We will showin a later section that the distribution of droplet sizes did notsignificantly affect the outcome of our dLAMP analysis.

FIG. 13E displays the reproducibility of sample digitization with 7 psiair pressure. For 15 individual chips, the average RVF and its standarddeviation were calculated. The average RVF varied within a 10% window,which demonstrates adequate reproducibility in chip performance. Alsothe standard deviation in droplet size was comparable for all chips,albeit with a somewhat larger chip-to-chip variation.

Droplet Stability at Elevated Temperatures

After filling, the chip was incubated at 65° C. for 70 minutes to carryout isothermal DNA amplification. At elevated temperatures,nanoliter-sized compartments are prone to water evaporation through thePDMS and partitioning of water into the oil phase. This would result inincreases in reagent concentration, for example the ionic strength,which might cause inhibition of the reaction.

We found the amount of sample evaporation during thermal incubation waslimited to roughly 10% of the digitized volume (FIG. 14A). Chamberslocated at the periphery of the chip were exposed to more bulk PDMS andshowed a slightly higher shrinkage as compared to chambers located inthe center of the array (see inlet images of FIG. 14A). The overalleffect, however, was small and we did not expect it to affect theamplification reaction. If the peripheral evaporation is still aconcern, the respective chambers could be ignored and only the innermostchambers could be considered for further analysis, as will be discussedbelow.

During incubation at 65° C., we expect isothermal DNA amplification toproceed in those compartments that contained one or more DNA templates.Positive amplification was evidenced by a large increase in Calceinfluorescence after pyrophosphate is released during the amplificationprocess. Those compartments that contained no template DNA should onlyshow background fluorescence. We next tested whether we could obtain adigital signature by amplification of a diluted sample of DNA (430-folddilution of the stock solution). FIG. 14B shows images and intensityscans across a section of a chip before and after incubation. Asexpected, some chambers showed a significant increase in fluorescence,while neighbouring chambers remained dark. Crosstalk between chambersthus appeared not to be an issue: LAMP-competent chambers were wellseparated by neighbouring chambers that showed only background signal.

Droplet Size Variation does not Affect dLAMP Analysis

Smaller droplets have a lower probability of containing one or moretemplate molecules; positive events are less likely to occur in smallerdroplets and should thus be weighted more in the analysis. To testwhether such a procedure would differ from results obtained bystraightforward counting of LAMP-active compartments, we compared thesetwo analysis schemes for three representative chips at three differentinitial template concentrations c_(i) (c_(i)=c₀/150 (A), c_(i)=c₀/430(B) and c_(i)=c₀/1300 (C)). Table 4 summarizes the results. In the firstcase, the fraction of LAMP-active chambers, f₀, was inferred from simplecounting of positive droplets, regardless of initial droplet size. Thiswas compared to an “effective” fraction, f_(eff), which we approximatedbased on the initial droplet volume. f_(eff) was calculated as the sumof the inverse relative volume fractions for all positive droplets. Inother words, a droplet that had a RVF of 50%, contributed twice as muchas a droplet that occupied the full side chamber. We then compared theratios of f₀ and f_(eff) between the three chips that were analysed. Wefound that for our system, both analysis schemes gave similar results inall parameters. The roughly 10% increase of f_(eff) over f₀ reflects the10-15% variation in initial droplet size. From this comparison, weconcluded that in our experiments the homogeneity of initial dropletsize was good enough to validate the straightforward counting method.

TABLE 4 Comparison of dLAMP analysis schemes f₀ f_(eff) F_(inner) (alldroplets) (all droplets) (inner droplets) c_(i) = c₀/150 (A) 0.309 0.3380.292 c_(i) = c₀/430 (B) 0.092 0.104 0.092 c_(i) = c₀/1300 (C) 0.0340.040 0.035 f_(X) ^(A)/f_(X) ^(B) 3.4 3.3 3.2 f_(X) ^(A)/f_(X) ^(C) 9.08.5 8.3 f_(X) ^(B)/f_(X) ^(C) 2.7 2.6 2.6

We also checked whether the preferential shrinkage of the peripheraldroplets affected the analysis. We compared the fraction ofLAMP-competent chambers for the case when: a) all droplets areconsidered in the analysis (f₀), and b) the peripheral droplets wereexcluded from the analysis (f_(inner)). Recall that the increasedshrinkage might increase local solute concentrations, which could affectthe amplification process. For all three concentrations considered, therelative fraction of LAMP-competent droplets did change by less than 6%,if only the innermost droplets were analysed. The respective ratios forthe different sample concentrations agreed to within 10% of each other.For dLAMP, we therefore considered all droplets in the analysis, thusavoiding unnecessary sample waste due to exclusion of the peripheraldroplets.

dLAMP Quantifies Relative and Absolute DNA Concentrations

An important application of digital DNA amplification is thequantification of absolute template concentration in a sample. We nextanalysed the performance of the dLAMP chip with a serial dilution of DNAtemplate in the sample. If on average less than one template is confinedin the droplet, then we expect the number of LAMP-competent chambers toscale approximately linearly with DNA concentration. The templateconsisted of the DNA control sample provided with the LoopAmp® kit. Wefirst performed UV absorption spectroscopy to estimate the templateconcentration in the DNA stock solution. The amount of DNA was too smallto provide a quantifiable absorption at 260 nm. We therefore attemptedDNA quantification using an intercalating dye. In this experiment, thetarget template was compared to a dilution series of λ-phage DNA ofknown concentration. All samples were incubated with a DNA intercalatingdye (Evagreen®, Biotium, Hayward, Calif., USA) for 30 minutes. Thefluorescence signal of each sample was then quantified on the Typhoon™imager. Since concentration and length of the λ-phage DNA were known(48502 bp), we obtained at least an accurate estimate of the number ofbp present in the template solution. The intensity from the unknownsample was comparable to the intensity from solutions of less than 10⁵λ-phage DNA per μl. Because of the low contrast in this concentrationregime, we could not narrow down the concentration further. Assuming theplasmid being comparable in size to a full λ-phage DNA, we thus estimatea concentration between 10⁴ and 10⁵ template copies per μl.

To determine the concentration from dLAMP experiments, we firstperformed dLAMP on a dilution series of template concentrations rangingfrom ¼ to 1/1300 of the stock solution. Each experiment was done atleast in triplicate. FIG. 15 shows representative images of severalchips after 70 minutes incubation at 65° C. FIG. 16 shows the respectivefraction of LAMP-competent chambers, f₀. To calculate f₀, we firstdetermined the number of initial chambers before incubation, We thencounted the number of amplified chambers, n_(a), which were chambersthat had at least a three-fold increase in fluorescence over backgroundas quantified from the image taken after incubation. f₀ was thencalculated as f₀=n_(a)/n_(i).

We observed a linear change in the number of LAMP-competent chamberswith DNA concentration for the 3 lowest concentrations analysed. Toestimate the concentration of target DNA via dLAMP, we computed alinearized Poisson fit to the three lowest sample concentrations. Thefit yielded a concentration of (0.99±0.03)×10⁴ copies per μl, which isin good agreement with our estimate from the DNA intercalationexperiment. This result confirmed that our dLAMP chip was able toaccurately reproduce relative changes in DNA concentration in a serialdilution of an unknown sample. Given the inaccuracy in the determinationof the original DNA concentration, however, the data did not necessarilydemonstrate our ability to determine absolute DNA concentrations withdLAMP. We therefore performed additional dLAMP experiments with a newtarget template of known concentration.

We chose the full λ-phage DNA template used for the intercalation assaywith a set of corresponding LAMP primers. The DNA concentration in thestock solution was measured via UV spectrophotometry to be 465 μg/ml,which corresponded to 8.9×10⁹ copies per μl. The sample was then diluteddown to 20 copies per μl in the final LAMP solution. For thisconcentration and a side chamber volume of 6 nanoliter, we expected toobserve amplification in about 12% of all chambers to occur. dLAMP wasperformed for 70 minutes and FIG. 16B shows images of a 535 well chipbefore and after the LAMP reaction. From the initial number of dropletsdiscretized on-chip (479) and the number of LAMP-competent chambersafter incubation (47) we computed a relative fraction of 9.8% ofpositive events. A second experiment in a 351 chamber array yielded acomparable fraction of 10.7% for the same template concentration. Thesmall difference between expected and measured values for f_(a) canprobably be attributed to pipetting errors accumulating over the 8-folddilution series.

We therefore concluded that our dLAMP chip was capable of correctlydetermining absolute DNA concentrations as well as relative changes inDNA concentrations. Together with its simple operation, dLAMP in a SDchip provides a convenient platform to perform isothermal, digital DNAquantification with minimum sample consumption.

We demonstrated successful on-chip loop-mediated DNA amplification in adigital format. Driven by the limits of existing platforms to performdigital DNA amplification in a simple-to-use format with minimum sampleconsumption, we refined our self-digitization concept to allow forisothermal DNA amplification at 65° C. Our method is simple and robust:once the aqueous phase is pipetted into the chip, the use of a simplepump head and constant air pressure is sufficient to induce samplediscretization without the need of further chip manipulation, such aspneumatic valving or mechanical action. Unlike most (if not all) otherreported digitization platforms, our design offers complete loss-lesssample compartmentalization. This is important in cases where sampleavailability is limited, such as point-of-care applications.

Digital LAMP signatures reproduced absolute DNA concentrations as wellas relative changes with sufficient accuracy within an incubation periodof 70 minutes. Droplet shrinkage during sample incubation at elevatedtemperatures was minimal and did not affect the amplification process.We also addressed the issue of variations in droplet size during chipfilling and demonstrated sufficient size homogeneity, with dropletvolumes varying around 10-15%. Differences in droplet volume canpotentially affect the average number of templates per droplet and theresulting digital signature. We verified that our autonomous chipfilling produced droplets with sufficiently homogeneous volumes so thatdata analysis simplifies to the counting of LAMP-competent chambers,without the need to consider the variation in droplet volume.

Our experimental protocol utilizes standard lab instrumentation forheating and imaging and as such is compatible with most diagnosticsettings, as no additional custom-built instrumentation is required.Because of the moderate reaction temperature of 65° C., chip incubationcould also be done in a thermal water bath, which would further simplifychip operation. Finally we note that, besides the increase in Calceinfluorescence, LAMP potentially generates a visual signal fromprecipitation of the pyrophosphate. Detection could be performed on asimple microscope, which would ultimately obviate the need forfluorescence equipment and reduce assay complexity even further.

Experimental Section on dLAMP

Chemicals and Reagents

The LoopAmp DNA® amplification kit and the Calcein fluorescenceindicator kit were purchased from SA Scientific (San Antonio, Tex.,USA). Positive control DNA and with a set of corresponding primers wasincluded in the kit. Light mineral oil, sorbitan monooleate (SPAN-80),bovine serum albumin (BSA), propylene glycol methyl ether acetate(PGMEA) and isopropyl alcohol were obtained from Sigma Aldrich (St.Louis, Mo., USA). Polydimethylsiloxane (PDMS, Sylgard 184 kit) waspurchased from Dow Corning (Midland, Mich., USA).

For the LAMP experiments on the full λ-phage DNA, the following set of 6primers was used (forward inner primer (FIP):5′-CAGCATCCCTTTCGGCATACCAGGTGGCAAGGGTAATGAGG-3′ (SEQ ID NO:1), backwardinner primer (BIP): 5′-GGAGGTTGAAGAACTGCGGCAGTCGATGGCGTTCGTACTC-3′ (SEQID NO:2), forward outer primer (F3): 5′-GAATGCCCGTTCTGCGAG-3′ (SEQ IDNO:3), backward outer primer (B3): 5′-TTCAGTTCCTGTGCGTCG-3′ (SEQ IDNO:4), loop forward primer (LF), 5′-GGCGGCAGAGTCATAAAGCA-3′ (SEQ IDNO:5), and loop backward primer (LB): 5′-GGCAGATCTCCAGCCAGGAACTA-3′ (SEQID NO:6). All primers were purchased from IDT (San Diego, Calif., USA).

Microfluidic Chip Fabrication

Microfluidic chips for digital LAMP were replicated inpolydimethylsiloxane (PDMS) with standard soft lithography. The networkof microfluidic channels and side chambers was designed in AutoCAD(Autodesk, San Rafael, Calif., USA) and printed onto a Mylar photomask(Fineline Imaging, Colorado Springs Colo., USA). The mask was used tofabricate a two layer SU-8 on-silicon master. For each layer, thefollowing steps were performed: SU-8 photoresist (SU-8 2050, MicroChem,Newton, Mass., USA) was spin coated onto a freshly cleaned siliconwafer. After soft baking, wafer and photomask were aligned and exposedto UV in a commercial mask aligner (Newport, Irvine Calif., USA). UVexposure led to crosslinking of SU-8 underneath the transparent areas ofthe photomask. After curing, non-exposed SU-8 was dissolved in PGMEA andthe wafer was cleaned with isopropyl alcohol and hard baked for 10minutes at 155° C. The height of the positive features in the master wasmeasured in a home-built interferometer to be around 75 μm. To avoidsticking of PDMS onto the wafer during replication, the wafer was coatedwith (tridecafluoro)-1,1,2,2-tetrahydrooctyl)trichlorosilane (Gelest,Morrisville, Pa., USA) by gas-phase deposition.

For chip replication, PDMS base and catalyst were mixed in a 10:1 weightratio as recommended by the manufacturer. The mix was degassed for 15-20minutes and spin coated onto the SU-8 on-silicon master to form a thinfilm, approximately 300 μm thick. After curing at 70° C. for 3 hours,the PDMS was peeled off the wafer. Access holes were punched into theelastomer with a sharpened 15 gauge punch. The PDMS replica was bondedto a microscope slide coated with a thin layer of cured PDMS via oxygenplasma treatment. The PDMS chip was stored at 115° C. for 2 days, whichreverted the surface back to hydrophobic.

Experimental Protocol

Prior to the experiment, a set of small troughs matching the inlet andoutlet holes was replicated into PDMS and attached to the chip withdouble-sided tape. Each trough provided a reservoir of approximately 50μl. The chip was placed under vacuum for 20-30 minutes to remove excessair from the bulk PDMS. 40 μl of light mineral oil supplemented with0.025% w/w SPAN-80 were placed into the inlet of the main channel toprime the chip. Air pressure was applied to maintain oil flow until theair was driven out of the chip. After priming, the chip was covered witha small piece of adhesive PCR sealant film (Bio-Rad, Hercules, Calif.,USA) to reduce water evaporation during the incubation.

13 μl of LAMP solution were freshly prepared following themanufacturer's protocol. The mix was supplemented with 1.2 g/l BSA tostabilise the polymerase during the reaction and 0.6 μl of theCalcein-based fluorescence detection kit. 1.8-2 μl of LAMP solution werepipetted into the inlet and formed an aqueous plug at the bottom of thetrough that was covered with excess oil. External air pressure wasapplied to move the aqueous plug through the channel network until thewhole sample became digitized on-chip. Digitization was visually checkedon a microscope (AZ 100, Nikon Instruments, Melville N.Y., USA). Thesacrificial water channel was manually filled with degased water usingnegative pressure on the outlet. All inlets were covered with at least20 μl of oil before the chip was incubated at 65° C. for 70 minutes on aThermocycler fitted with in situ adaptor (Mastercycler, Eppendorf,Westbury, N.Y., USA).

Chip Imaging and Quantification

Before and after thermal incubation, all chips were scanned on avariable mode imager (Typhoon FLA9000, GE Healthcare, Pittsburgh, Pa.,USA). The Calcein fluorescence inside the chambers was excited at 473 nmand images were taken through a long pass filter (510LP) with 10 μmpixel resolution and a voltage of 350V applied to the photomultipliertube. Subsequent image analysis was performed in ImageJ (rsbweb.nih.gov)to quantify the number and size distribution of discretized volumes aswell as the number of chambers that showed DNA amplification. Furtherdata analysis was done with IGOR Pro (WaveMetrics, Lake Oswego, Oreg.,USA). Although initially the Calcein fluorescence was highly quenched,its residual emission could still be monitored with the Typhoon imagerto determine initial droplet size in the chamber. The distribution ofinitial droplet sizes was quantified in terms of the retained volumefraction (RVF) estimated as the ratio of the droplet area to the area ofthe side chamber. A threshold equal to ⅓ of the average pixel intensityof the Calcein fluorescence in droplets was used to discriminate againstbackground. Only droplets with a RVF of at least 0.15 were consideredfor further analysis.

As provided herein, a statistical analysis of a digital droplet assayusing droplets from a continuous distribution of sizes is presented. Thebest fit concentrations are moderately insensitive to modest unbiasederrors in the determination of the individual droplet volumes, so therequired accuracy of that determination should not be an unreasonableburden. A simple method for estimating the confidence when comparingresults from different samples is presented. It produces estimates ofthe confidence in reasonable agreement with a more computationallyintensive estimate of the confidence. From simulations, the statisticalpower of the method can be calculated for three different distributionsof the droplet diameters for 1.2 and 1.5 fold resolutions. Given adesired resolution, droplet size distribution and number of droplets,these methods can be used to determine the range of concentrations forwhich this method should yield usable results.

While the present invention has been described with an emphasis oncertain embodiments, it will be apparent to those skilled in the artthat various modifications and variations can be made in the presentinvention without departing from the scope or spirit of the invention.Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope and spiritof the invention being defined by the following claims.

What is claimed is:
 1. A system for using digital measurements todetermine a concentration of a sample, the system comprising: a sampleholder containing a plurality of droplets having a volume distributionwherein the volume distribution is a continuous volume distribution; adetector for detecting a detectable agent contained in at least onedroplet of the plurality; and a computer comprising a memory device withexecutable instructions stored thereon, the instructions, when executedby a processor, cause the processor to: analyze a portion of theplurality of droplets to determine individual volumes of the droplets inthe portion of the plurality and a number of droplets in the portion ofthe plurality that contain a detectable agent; and use individualvolumes of the droplets in the portion of the plurality and the numberof droplets in the portion of the plurality that contain the detectableagent to determine the concentration of the sample.
 2. The system ofclaim 1, wherein the plurality of droplets is produced in an emulsion bycombining immiscible fluids.
 3. The system of claim 2, wherein theimmiscible fluids comprise water and oil.
 4. The system of claim 1,wherein the detectable agent is fluorescent.
 5. The system of claim 4,wherein the detectable agent is associated with a nucleic acid molecule,a peptide, a protein, or a combination thereof.
 6. The system of claim1, wherein at least one of the droplets in the plurality comprises anamplified product from a polymerase chain reaction (PCR), rolling circleamplification (RCA), nucleic acid sequence based amplification (NASBA),loop-mediated amplification (LAMP), or a combination thereof.
 7. Thesystem of claim 1, wherein the concentration of the detectable agent isdetermined over a dynamic range of at least three orders of magnitude.8. The system of claim 1, wherein the concentration of the detectableagent is determined over a dynamic range of at least six orders ofmagnitude.
 9. The system of claim 1, wherein the volumes in the volumedistribution vary by more than a factor of
 2. 10. The system of claim 1,wherein the volumes in the volume distribution vary by more than afactor of 10 or by more than a factor of 100.